WO2013059530A2 - Peptidomimetic macrocycles - Google Patents

Peptidomimetic macrocycles Download PDF

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Publication number
WO2013059530A2
WO2013059530A2 PCT/US2012/060919 US2012060919W WO2013059530A2 WO 2013059530 A2 WO2013059530 A2 WO 2013059530A2 US 2012060919 W US2012060919 W US 2012060919W WO 2013059530 A2 WO2013059530 A2 WO 2013059530A2
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WIPO (PCT)
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amino acid
peptidomimetic macrocycle
macrocycle
hiv
independently
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PCT/US2012/060919
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French (fr)
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WO2013059530A3 (en
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Noriyuki Kawahata
Carl Elkin
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Aileron Therapeutics, Inc.
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Publication of WO2013059530A2 publication Critical patent/WO2013059530A2/en
Publication of WO2013059530A3 publication Critical patent/WO2013059530A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Entry of HIV- 1 into cells is an ordered, multi-step process leading to fusion of viral and cell membranes.
  • the first step in entry is binding of the viral envelope to CD4 on the surface of macrophages or T-helper lymphocytes. Binding to CD4 is mediated by gpl20, the surface subunit of the envelope.
  • the envelope glycoprotein In its native form, the envelope glycoprotein is a heterotrimer of three gpl20 and three HIV HIV gp41 molecules. Conformational changes triggered by CD4 binding, and other structural alterations, allows the N-terminus of HIV gp41 to insert into the target cell membrane, resulting in fusion and viral infection.
  • Enfuvirtide (FUZEON®, T-20), the first HIV entry inhibitor, is a 36-mer synthetic oligopeptide derived from the HR2 region of the HIV-1 envelope HIV gp41 subunit. Binding of enfuvirtide to HIV gp41 prevents the formation of a six -helix bundle between the HR1 and HR2 helices of three HIV gp41 proteins. This HIV HIV gp41 six- helix bundle is required for proper HIV fusion with the target cell, and antagonism of the fusion complex formation blocks virus entry and prevents propagation of wild-type and resistant strains. Because the drug is an oligopeptide, it is administered subcutaneous ly by injection.
  • the present invention provides HIV gp41 -derived peptidomimetic macrocycles that are designed to possess improved pharmaceutical properties relative to enfuvirtide. These improved properties include enhanced chemical and in vivo stability, increased potency and reduced immunogenicity ⁇ i.e., fewer or less severe injection site reactions). These peptidomimetic macrocycles are useful for the prevention and treatment of HIV infection and resulting immunedeficiencies ⁇ i.e., AIDS).
  • a cross-linked polypeptide described herein can have improved biological activity relative to a corresponding polypeptide that is not cross-linked.
  • the HIV HIV gp41 peptidomimetic macrocycles are thought to interfere with binding of HIV gp41 HR1 and HR2 helices to each other to prevent assembly of the active six-helix bundle fusion complex, thereby inhibiting entry of the HIV virus.
  • the HIV HIV gp41 peptidomimetic macrocycles described herein can be used therapeutically, for example, to prevent or to treat HIV infection and resulting immunedeficiencies ⁇ i.e., AIDS).
  • a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1, 2, 3, and 4, wherein the
  • peptidomimetic macrocycle has an IC 50 of about 10000 nM or less against a target and in cells selected from the group consisting of: target: HIV-1 IIIB, cells: CEM-SS; target: HIV-1NL4-3, cells: MT-4; and target: HIV-1MDR769, cells: hPBMC, wherein the peptidomimetic macrocycle is not a compound having the sequence:
  • a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to HIV gp41626-663, further comprising at least two independent crosslinkers, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid, wherein the first amino acid and the second amino acid are selected from the group consisting of amino acids: 629 and 633; 630 and 634; 632 and 636; and 633 and 637; wherein the third amino acid and the fourth amino acid are selected from the group consisting of amino acids: 650 and 654; 651 and 655; 652 and 656; 653 and 657; 654 and 658; 655 and 659; 656 and 660; and 657 and 771;with the proviso that when the first crosslinker connects amino acids pairs 629 and 633 and the second crosslinker connects amino acid pair 653 and 657, there is an amino acid sequence which is at least about
  • sequence which is at least about 60% identical to HIV gp41 638-673, further having one crosslinker, wherein the crosslinker connects a first amino acid to a second amino acid, wherein the first amino acid and the second amino acid are 652 and 659.
  • a peptidomimetic macrocycle which is at least about 60% identical to HIV gp41 638-673, further comprising at at least two independent crosslinkers, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid, wherein the amino acid pairs are selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671; wherein when the first crosslinker connects amino acid pair 640 and 644, the second crosslinker does not connect the amino acid pair 664 and 668 or 666 and 670; and
  • associated immunodeficiencies i.e., AIDS
  • an antagonist of the six -helix bundle HIV HIV gp41 fusion complex wherein the antagonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
  • Also provided herein is a method of preventing or treating HIV infection and associated immunodeficiencies (i.e., AIDS) in a subject by administering an antagonist which can be a peptidomimetic macrocycle herein of the six -helix bundle HIV HIV gp41 fusion complex, wherein the frequency or intensity of the site of injection reaction is reduced relative to that of enfuvirtide.
  • an antagonist which can be a peptidomimetic macrocycle herein of the six -helix bundle HIV HIV gp41 fusion complex, wherein the frequency or intensity of the site of injection reaction is reduced relative to that of enfuvirtide.
  • HIV infection and associated immunodeficiencies i.e., AIDS
  • a method of preventing or treating HIV infection and associated immunodeficiencies comprising administering to the subject a peptidomimetic macrocycle of the invention.
  • Also provided is a method of inhibiting HIV viral entry in a subject comprising
  • a peptidomimetic macrocycle of the invention or a method of antagonizing the formation of the six -helix bundle HIV HIV gp41 fusion complex in a subject comprising administering to the subject such a peptidomimetic macrocycle herein.
  • FIGURE 1 shows improved stabilities to trypsin proteolysis of the peptidomimetic
  • FIGURE 2 shows in vitro anti-viral activities of the peptidomimetic macrocycles of the invention.
  • FIGURE 3 shows in vitro anti-viral activities of the peptidomimetic macrocycles of the invention
  • ranges include the range endpoints. Additionally, every subrange and value within the range is present as if explicitly written out.
  • microcycle refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.
  • peptidomimetic macrocycle or “crosslinked polypeptide” refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule.
  • Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the a carbon of the first amino acid residue (or analog) to the a carbon of the second amino acid residue (or analog).
  • the peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle.
  • a "corresponding uncrosslinked polypeptide" when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle.
  • Peptidomimetic macrocycles include, for example: macrocycles having one crosslinker (e.g., stapled peptidomimetic macrocycles); macrocycles having two or more crosslinkers that are not in tandem (e.g., multiply stapled peptidomimetic macrocycles); macrocycles having two crosslinkers that are in tandem (e.g. , stitched peptidomimetic macrocycles); and macrocycles that are stitched and further stapled with at least one additional crosslinker.
  • crosslinker e.g., stapled peptidomimetic macrocycles
  • macrocycles having two or more crosslinkers that are not in tandem e.g., multiply stapled peptidomimetic macrocycles
  • macrocycles having two crosslinkers that are in tandem e.g. , stitched peptidomimetic macrocycles
  • macrocycles that are stitched and further stapled with at least one additional crosslinker e.g., stitched peptidomim
  • the term “stability” refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle of the invention as measured by circular dichroism, NMR or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo.
  • Non-limiting examples of secondary structures contemplated in this invention are a-helices, ⁇ -turns, and ⁇ -pleated sheets.
  • the term “helical stability” refers to the maintenance of a helical structure by a peptidomimetic macrocycle of the invention as measured by circular dichroism or NMR.
  • the peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in a-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.
  • amino acid refers to a molecule containing both an amino group and a
  • Suitable amino acids include, without limitation, both the D-and L- isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes.
  • amino acid as used herein, includes without limitation, a-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.
  • a-amino acid refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the a-carbon.
  • ⁇ -amino acid refers to a molecule containing both an amino group and a carboxyl group in a ⁇ configuration.
  • Naturally occurring amino acid refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
  • amino acid analog refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle.
  • Amino acid analogs include, without limitation, ⁇ -amino acids and amino acids where the amino or carboxy group is substituted by a similarly reactive group (e.g. , substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).
  • non-natural amino acid refers to an amino acid which is not one of the the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
  • a "non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially abolishing its essential biological or biochemical activity (e.g., receptor binding or activation).
  • An "essential” amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., K, R, H
  • acidic side chains e.g., D, E
  • uncharged polar side chains e.g., G, N, Q, S, T, Y, C
  • nonpolar side chains e.g., A, V, L, I, P, F, M, W
  • beta-branched side chains e.g., T, V, I
  • aromatic side chains e.g., Y, F, W, H.
  • a predicted nonessential amino acid residue in a polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine).
  • member refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms.
  • cyclodecane, 1 ,2-difluoro-decane and 1,3- dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.
  • amino acid side chain refers to a moiety attached to the a-carbon in an amino acid.
  • amino acid side chain for alanine is methyl
  • amino acid side chain for phenylalanine is phenylmethyl
  • amino acid side chain for cysteine is thiomethyl
  • amino acid side chain for aspartate is carboxymethyl
  • amino acid side chain for tyrosine is 4-hydroxyphenylmethyl
  • Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically (e.g., an ⁇ , ⁇ di-substituted amino acid).
  • ⁇ , ⁇ di-substituted amino acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the a-carbon) that is attached to two natural or non-natural amino acid side chains.
  • polypeptide encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments).
  • macrocyclization reagent or “macrocycle-forming reagent” as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle of the invention by mediating the reaction between two reactive groups.
  • Reactive groups may be, for example, an azide and alkyne
  • macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, Cul or CuOTf, as well as Cu(II) salts such as Cu(C0 2 CH 3 ) 2 , CuS0 4 , and CuCl 2 that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate.
  • a reducing agent such as ascorbic acid or sodium ascorbate
  • Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh3) 2 , [Cp*RuCl] 4 or other Ru reagents which may provide a reactive Ru(II) species.
  • the reactive groups are terminal olefins. In such embodiments, the
  • macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts.
  • metathesis catalysts include Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and
  • the reactive groups are thiol groups.
  • the macrocyclization reagent is, for example, a linker
  • halo or halogen refers to fluorine, chlorine, bromine or iodine or a radical thereof.
  • alkyl refers to a hydrocarbon chain that is a straight chain or branched chain, containing the indicated number of carbon atoms.
  • Ci-Cio indicates that the group has from 1 to 10 (inclusive) carbon atoms in it.
  • alkyl is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it.
  • Alkyl groups can have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.
  • Alkyl groups include, for example, methyl, ethyl, n-propyl, iso-propyl, and butyl groups.
  • alkylene refers to a divalent alkyl (i.e., -R-).
  • alkenyl refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds.
  • the alkenyl moiety contains the indicated number of carbon atoms. For example, C 2 -Cio indicates that the group has from 2 to 10 (inclusive) carbon atoms in it.
  • lower alkenyl refers to a C 2 -C6 alkenyl chain. In the absence of any numerical designation, "alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
  • alkynyl refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds.
  • the alkynyl moiety contains the indicated number of carbon atoms.
  • C2-C10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it.
  • lower alkynyl refers to a C 2 -C6 alkynyl chain.
  • alkynyl is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
  • aryl refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent.
  • aryl groups include phenyl, naphthyl and the like.
  • Arylalkyl refers to an aryl group substituted with an aryl.
  • one of the aryl group's hydrogen atoms has been replaced with a C1-C5 alkyl group, as defined above.
  • Representative examples of an arylalkyl group include, but are not limited to, 2- methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4- ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3- butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2- isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3- isobutylphenyl, 4-
  • Arylamido refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more -C(0)NH 2 groups.
  • Representative examples of an arylamido group include 2-C(0)NH2 -phenyl, 3-C(0)NH 2 -phenyl, 4- C(0)NH 2 -phenyl, 2-C(0)NH 2 -pyridyl, 3-C(0)NH 2 -pyridyl, and 4-C(0)NH 2 -pyridyl,
  • Alkylheterocycle refers to a C1-C5 alkyl group, as defined above, wherein one of the
  • C1-C5 alkyl group's hydrogen atoms has been replaced with a heterocycle.
  • Representative examples of an alkylheterocycle group include, but are not limited to, -CH 2 CH 2 - morpholine, -CH 2 CH 2 -piperidine, -CH 2 CH 2 CH 2 -morpholine, and -CH 2 CH 2 CH 2 - imidazole.
  • Alkylamido refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a -C(0)NH 2 group.
  • alkanol refers to a C 1 -C5 alkyl group, as defined above, wherein one of the C 1 -C5 alkyl group's hydrogen atoms has been replaced with a hydroxyl group.
  • Representative examples of an alkanol group include, but are not limited to, -CH 2 OH, -CH 2 CH 2 OH, - CH 2 CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 OH, -CH 2 CH 2 CH 2 CH 2 CH 2 OH, -CH 2 CH(OH)CH 3 , - CH 2 CH(OH)CH 2 CH 3 , -CH(OH)CH 3 and -C(CH 3 ) 2 CH 2 OH.
  • Alkylcarboxy refers to a C 1 -C5 alkyl group, as defined above, wherein one of the C 1 -C5 alkyl group's hydrogen atoms has been replaced with a --COOH group.
  • alkylcarboxy group examples include, but are not limited to, -CH 2 COOH, - CH 2 CH 2 COOH, -CH 2 CH 2 CH 2 COOH, -CH 2 CH 2 CH 2 CH 2 COOH, -CH 2 CH(COOH)CH 3 , - CH 2 CH 2 CH 2 CH 2 COOH, -CH 2 CH(COOH)CH 2 CH 3 , -CH(COOH)CH 2 CH 3 and - C(CH 3 ) 2 CH 2 COOH.
  • cycloalkyl as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted.
  • Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclop entenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1 -3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2, 3, or 4 atoms of each ring are substituted by a substituent.
  • heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.
  • heteroarylalkyl or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl.
  • heteroarylalkoxy refers to an alkoxy substituted with heteroaryl.
  • heterocyclyl refers to a nonaromatic 5-8 membered monocyclic, 8-12
  • heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
  • substituted refers to a group replacing a second atom or group such as a
  • substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
  • the compounds of this invention contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included in the present invention unless expressly provided otherwise.
  • the compounds of this invention are also represented in multiple tautomeric forms, in such instances, the invention includes all tautomeric forms of the compounds described herein (e.g. , if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All such isomeric forms of such compounds are included in the present invention unless expressly provided otherwise. All crystal forms of the compounds described herein are included in the present invention unless expressly provided otherwise.
  • on average represents the mean value derived from performing at least three independent replicates for each data point.
  • biological activity encompasses structural and functional properties of a macrocycle of the invention.
  • Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell
  • the peptide sequences are derived from the HIV HIV gp41
  • a non-limiting exemplary list of suitable HIV HIV gp41 peptides for use in the present invention is given in Table 1 and Table 2 below (X residues form cross-links to one other X residue, Z residues form cross-links to one other Z residue, and XX residues form cross-links with two other X residues):
  • a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1, 2 and 3, wherein the peptidomimetic macrocycle has an IC 50 of about 10000 nM or less against a target and in cells selected from the group consisting of: target: HIV-1 IIIB, cells: CEM-SS; target: HIV-1NL4-3, cells: MT-4; and target: HIV-1MDR769, cells: hPBMC, wherein the peptidomimetic macrocycle is not a compound having the sequence:
  • HIV gp41 is discussed, for example, in International Application Publication No. WO 2009/018261.
  • the sequence of the peptidomimetic macrocycle can be, for example, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1 , 2 and 3.
  • the sequence of the peptidomimetic macrocycle can be selected from the group consisting of the amino acid sequences in Tables 1 , 2, and 3.
  • the target can be HIV-1 IIIB and cells can be CEM-SS.
  • the target can be HIV-1NL4-3 and cells can be MT-4.
  • the target can be HIV- 1 MDR769 and the cells can be hPBMC .
  • the peptidomimetic macrocycle can have an IC 50 , for example, of about 10000 nM or less, about 6000 nM or less, about 3000 nM or less, about 1000 nM or less, about 500 nM or less, about 100 nM or less, about 10 nM or less, about 5 nM or less, about 4 nM or less, about 3nM or less, about 2 nM or less, about 1 nM or less, or about 0.5 nM or less, against a target and in cells selected from the group consisting of target: HIV-1 IIIB, cells: CEM-SS; target: HIV-1NL4-3, cells: MT-4; and target: HIV-1MDR769, cells: hPBMC.
  • IC 50 for example, of about 10000 nM or less, about 6000 nM or less, about 3000 nM or less, about 1000 nM or less, about 500 nM or less, about 100 nM or less, about 10 nM or
  • the peptidomimetic macrocycle can have an IC 50 of, for example, about 10 nM or less, about 5 nM or less, about 4 nM or less, about 3nM or less, about 2 nM or less, about 1 nM or less, or about 0.5 nM or less, when the target is HIV-1MDR769 and the cells are hPBMC.
  • the peptidomimetic macrocycle can have a positive number of amino acids, for example, of 60 or less, or 50 or less, of 40 or less, of 30 or less, or of 20 of less.
  • the peptidomimetic macrocycle can comprise one or more independent crosslinkers, each crosslinker independently linking the a-positions of two amino acids of the peptidomimetic macrocycle.
  • At least one of the two amino acids can be an ⁇ , ⁇ - disubstituted amino acid.
  • the peptidomimetic macrocycle can comprise one crosslinker linking the ⁇ -positions of two amino acids of the peptidomimetic macrocycle.
  • the peptidomimetic macrocycle can comprise two independent crosslinkers, each
  • crosslinker independently linking the ⁇ -positions of two amino acids of the
  • a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to HIV gp41626-663, further comprising at least two independent crosslinkers, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid, wherein the first amino acid and the second amino acid are selected from the group consisting of amino acids: 629 and 633; 630 and 634; 632 and 636; and 633 and 637; wherein the third amino acid and the fourth amino acid are selected from the group consisting of amino acids: 650 and 654; 651 and 655; 652 and 656; 653 and 657; 654 and 658; 655 and 659; 656 and 660; and 657 and 771; with the proviso that when the first crosslinker connects amino acids pairs 629 and 633 and the second crosslinker connects amino
  • the peptidomimetic macrocycle can be at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to HIV gp41626-663.
  • the peptidomimetic macrocycle can comprise an additional cross linker, wherein the additional crosslinker connects an amino acid pair selected from the group consisting of: 640 and 644; 641 and 645; and 642 and 646.
  • the first crosslinker can connect amino acid pair 629 and 633, and the second crosslinker can connect amino acid pair 654 and 658.
  • the peptidomimetic macrocycle can have a sequence that is at least about 60% identical to the sequence:
  • the sequence can be at least about 60%>, at least about 70%>, at least about 80%>, at least about 85%o, at least about 90%>, or at least about 95% identical to SP-36.
  • the first crosslinker connect amino acid pair 629 and
  • the peptidomimetic macrocycle can have a sequence that is at least about 60% identical to the sequence:
  • the sequence can be at least about 60%>, at least about 70%>, at least about 80%>, at least about 85%o, at least about 90%>, or at least about 95% identical to SP-37.
  • the first crosslinker can connect amino acid pair 633 and 637
  • the second crosslinker can connect amino acid pair 655 and 659.
  • the peptidomimetic macrocycle can have a sequence that is at least about 60% identical to the sequence:
  • the sequence can be at least about 60%>, at least about 70%>, at least about 80%>, at least about 85%o, at least about 90%>, or at least about 95% identical to SP-34.
  • the peptidomimetic macrocycle can comprise a helix.
  • the peptidomimetic macrocycle can comprise an alpha-helix.
  • the peptidomimetic macrocycle can comprise an ⁇ , ⁇ -disubstituted amino acid.
  • the peptidomimetic macrocycle can be such that at least one of the two amino acids in one or more amino acid pairs is an ⁇ , ⁇ -disubstituted amino acid.
  • a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60%> identical to HIV gp41 638-673, further having one crosslinker, wherein the crosslinker connects a first amino acid to a second amino acid, wherein the first amino acid and the second amino acid are 652 and 659.
  • peptidomimetic can be, for example, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% 60% identical to HIV gp41 638-673.
  • the peptidomimetic macrocycle can comprise a helix.
  • the peptidomimetic macrocycle can comprise an alpha-helix.
  • the peptidomimetic macrocycle can comprise an ⁇ , ⁇ -disubstituted amino acid.
  • the peptidomimetic macrocycle can be such that at least one of the two amino acids in one or more amino acid pairs is an ⁇ , ⁇ -disubstituted amino acid.
  • first crosslmker connects a first amino acid to a second amino acid
  • second crosslmker connects a third amino acid to a fourth amino acid
  • each pair of first and second amino acids are selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671;wherein when the first crosslmker connects amino acid pair 640 and 644, the second crosslmker does not connect the amino acid pair 664 and 668 or 666 and 670; and wherein when the first crosslmker connects amino
  • the peptidomimetic macrocycle can be at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to HIV gp41 638-673.
  • the first crosslmker can connect an amino acid pair selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; and 653 and 657.
  • the second crosslmker can connecxt an amino acid pair selected from the group consisting of: 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671.
  • the peptidomimetic macrocycle can comprise a helix.
  • the peptidomimetic macrocycle can comprise an alpha-helix.
  • the peptidomimetic macrocycle can comprise an ⁇ , ⁇ -disubstituted amino acid.
  • the peptidomimetic macrocycle can be such that at least one of the two amino acids in one or more amino acid pairs is an ⁇ , ⁇ -disubstituted amino acid.
  • the peptidomimetic macrocycle can be of Formula (I):
  • each A, C, D, and E is independently a natural or non-natural amino acid
  • B is a natural or non-natural amino acid, amino acid analog, , [-NH-L3-
  • Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is unsubstituted or substituted with halo-;
  • R 3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
  • heterocycloalkyl cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which except hydrogen is optionally substituted with R 5 ;
  • L is a macrocycle-forming linker of the formula -Li-L 2 -;
  • Li and L 2 and L 3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4 -K-R 4 -] n , each being optionally substituted with R 5 ;
  • each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
  • heterocycloalkylene arylene, or heteroarylene
  • each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
  • each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 ) 2 , -SR 6 , -SOR5, -S0 2 R6, -C0 2 R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R 5 , or part of a cyclic structure with a D residue;
  • R8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R 5 , or part of a cyclic structure with an E residue;
  • v and w are independently integers from 1-1000, for example: 1-500, 1-300, 1-200, 1- 100, 1-50, 1-40, 1-30, 1-20, 1- 10;
  • u is an integer from 1-10, for example 1, 2, or 3;
  • x, y and z are independently integers from 0-10; for example, the sum of x+y+z can equal 2, 3 or 6, for example, 3 or 6; and
  • n is an integer from 1-5.
  • At least one of Ri and R 2 is alkyl, unsubstituted or substituted with halo-.
  • both Ri and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of Ri and R 2 is methyl. In other embodiments, Ri and R 2 are methyl.
  • x+y+z is at least 3. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
  • a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gin-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
  • each compound of the invention may encompass peptidomimetic macrocycles which are the same or different.
  • a compound of the invention may comprise
  • the peptidomimetic macrocycle of the invention comprises a
  • At least one of A, B, C, D or E is an ⁇ , ⁇ -disubstituted amino acid.
  • B is an ⁇ , ⁇ -disubstituted amino acid.
  • at least one of A, B, C, D or E is 2-aminoisobutyric acid.
  • the length of the macrocycle-forming linker L as measured from a first Ca to a second Ca is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Ca to a second Ca.
  • the peptidomimetic macrocycle of Formula (I) is:
  • each Ri and R 2 is independently independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
  • the peptidomimetic macrocycle of Formula (I) is:
  • the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below:
  • AA represents any natural or non-natural amino acid side chain and " is [D] v , [E] v as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In other embodiments, n is less than 50.
  • R H, alkyl, other substituent
  • L is a macrocycle-forming linker of the formula
  • D and/or E in the compound of Formula I are further modified in order to facilitate cellular uptake.
  • lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.
  • a peptidomimetic macrocycle represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers.
  • a peptidomimetic macrocycle comprises two macrocycle-forming linkers.
  • linkers described herein may be used in any combination with any of the sequences shown in Tables 1-6 and also with any of the R- substituents indicated herein.
  • the peptidomimetic macrocycle comprises at least one a-helix motif.
  • A, B and/or C in the compound of Formula I include one or more a- helices.
  • a-helices include between 3 and 4 amino acid residues per turn.
  • the a-helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3 to 20 amino acid residues.
  • the a-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns.
  • the length of the macrocycle-forming linker L from a first Ca to a second Ca is selected to increase the stability of an a-helix.
  • the macrocycle-forming linker spans from 1 turn to 5 turns of the a-helix.
  • the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the a-helix.
  • the length of the macrocycle-forming linker is approximately 5 A to 9 A per turn of the a-helix, or approximately 6 A to 8 A per turn of the a-helix.
  • the length is equal to approximately 5 carbon- carbon bonds to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds.
  • the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds.
  • the macrocycle-forming linker spans approximately 3 turns of an a-helix, the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds.
  • the linkage contains
  • the linkage contains approximately 7 atoms to 15 atoms
  • the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms.
  • the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms.
  • the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms.
  • the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members.
  • the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members.
  • the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members.
  • the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members.
  • the macrocycle-forming linker spans approximately 5 turns of the a-helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.
  • the peptidomimetic macrocycle is of Formula (II) or (Ha):
  • each A, C, D, and E is independently a natural or non-natural amino acid
  • B is a natural or non-natural amino acid, amino acid analog, O , [-NH-L3-
  • Ri and R 2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;
  • R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
  • heterocycloalkyl cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which except hydrogen is optionally substituted with R 5 ;
  • Li and L 2 and L3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R4-] n , each being optionally substituted with R 5 ;
  • each R 4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
  • heterocycloalkylene arylene, or heteroarylene
  • each K is O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ; each R 5 is independently halogen, alkyl, -OR 6 , -N(R 6 )2, -SR 6 , -SOR5, -S0 2 R6, -C0 2 R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • each R 6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
  • R 7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R 5 ;
  • v and w are independently integers from 1-1000;
  • u is an integer from 1-10, for example 1, 2, or 3;
  • x, y and z are independently integers from 0-10; for example, the sum of x+y+z can equal
  • n is an integer from 1-5.
  • At least one of Ri and R 2 is alkyl, unsubstituted or substituted with halo-.
  • both Ri and R 2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of Ri and R 2 is methyl. In other embodiments, Ri and R 2 are methyl.
  • x+y+z is at least 1. In other embodiments of the invention, x+y+z is at least 2. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected.
  • a sequence represented by the formula [A] x when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gin-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
  • the peptidomimetic macrocycle of the invention comprises a
  • At least one of A, B, C, D or E is an ⁇ , ⁇ - disubstituted amino acid.
  • B is an ⁇ , ⁇ -disubstituted amino acid.
  • at least one of A, B, C, D or E is 2-aminoisobutyric acid.
  • At least one of A, B, C, D or E is .
  • the length of the macrocycle-forming linker L as measured from a first Ca to a second Ca is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Ca to a second Ca.
  • R H, alkyl, other substituent
  • L is a macrocycle-forming linker of the formula
  • the peptidomimetic macrocycle can have the Formula III):
  • each A, C, D, and E is independently an amino acid
  • B is an amino acid, , [-NH-L3-CO-], [-NH-L3-SO 2 -], or [-NH-L3-];
  • L' is a macrocycle-forming linker of the formula -Li'-L 2 '-;
  • Ri' and R 2 ' are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
  • Li ' and L 2 ' are independently alkylene, alkenylene, alkynyl ene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R 4 -K-R 4 -] n , each being optionally substituted with R 5 ;
  • each K is independently O, S, SO, S0 2 , CO, C0 2 , or CONR 3 ;
  • R 7 ' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue;
  • Rs' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue;
  • v' and w' are independently integers from 1-100;
  • x', y' and z' are independently integers from 0-10;
  • n is an integer from 1-5.
  • the present invention provides novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled.
  • labeled peptidomimetic macrocycles based on HIV gp41 can be used in a binding assay along with small molecules that competitively bind to the HIV gp41 six helix bundle assembly or sub-complexes thereof.
  • Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the HIV gp41 system. Such binding studies may be performed with any of the peptidomimetic macrocycles disclosed herein and their binding partners.
  • the invention further provides for the generation of antibodies against the
  • these antibodies specifically bind both the peptidomimetic macrocycle and the precursor peptides, such as HIV gp41, to which the peptidomimetic macrocycles are related.
  • Such antibodies for example, disrupt the native protein-protein interactions, for example, in the HIV gp41 fusion complex.
  • Also provided is a method of preventing HIV infection in a subject comprising
  • a method of inhibiting HIV viral entry in a subject comprising administering to the subject a peptidomimetic macrocycle herein.
  • immunodeficiencies in a subject comprising administering to the subject a
  • Also provided is a method of inhibiting HIV viral entry in a subject comprising
  • HIV HIV gp41 fusion complex in a subject comprising administering to the subject a peptidomimetic macrocycle herein.
  • a method of preventing or treating HIV infection and associated immunodeficiencies comprising administering an antagonist of the six -helix bundle HIV HIV gp41 fusion complex, wherein the antagonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week, wherein the antagonist can be a peptidomimetic macrocycle herein.
  • immunodeficiencies i.e., AIDS
  • AIDS immunodeficiencies
  • the antagonist can be any peptidomimetic macrocycle herein.
  • the subject can be a subject in need thereof (e.g., in need of treatment or prevention or antagonizing). Any method herein can comprise administering an effective amount of a peptidomimetic macrocyle herein or compound of the invention herein to a warm blooded animal, including a human.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with HIV infection.
  • the present invention provides methods for treating or preventing a disease including HIV infection and associated immunodeficiencies (i.e., AIDS) by interfering with the assembly of the six helix bundle HIV gp41 fusion complex.
  • a disease including HIV infection and associated immunodeficiencies (i.e., AIDS)
  • treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
  • Peptidomimetic macrocycles of the invention may be prepared by any of a variety of methods known in the art.
  • any of the residues indicated by "X" in Tables 1, 2 or 3 may be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.
  • peptidomimetic macrocycles of Formula I For example, the preparation of peptidomimetic macrocycles of Formula I is described in Schafmeister et al, J. Am. Chem. Soc. 122:5891-5892 (2000); Schafffle & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al, Science 305: 1466-1470 (2004); US Patent No. 7,192,713 and PCT application WO 2008/121767.
  • the ⁇ , ⁇ -disubstituted amino acids and amino acid precursors disclosed in the cited references may be employed in synthesis of the peptidomimetic macrocycle precursor polypeptides.
  • the "S5-olefm amino acid” is (S)-a-(2'-pentenyl) alanine and the "R8 olefin amino acid” is (R)-a-(2'-octenyl) alanine.
  • the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle.
  • the peptidomimetic macrocyles of the invention are of Formula II or Ila. Methods for the preparation of such macrocycles are described, for example, in US Patent No. 7,202,332.
  • Additional methods of forming peptidomimetic macrocycles which are envisioned as suitable to perform the present invention include those disclosed by Mustapa, M. Firouz Mohd et al, J. Org. Chem (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp. 1403-1406; U.S. Patent No. 5,364,851 ; U.S. Patent No. 5,446, 128; U.S. Patent No. 5,824,483; U.S. Patent No. 6,713,280; and U.S. Patent No. 7,202,332.
  • aminoacid precursors are used containing an additional substituent R- at the alpha position. Such aminoacids are incorporated into the macrocycle precursor at the desired positions, which may be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then performed according to the indicated method.
  • peptidomimetic macrocycle of the invention has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.
  • the secondary structure of polypeptides with a-helical domains will reach a dynamic equilibrium between random coil structures and ⁇ -helical structures, often expressed as a "percent helicity".
  • alpha-helical domains are predominantly random coils in solution, with ⁇ -helical content usually under 25%.
  • Peptidomimetic macrocycles with optimized linkers possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide.
  • macrocycles of the invention will possess an alpha-helicity of greater than 50%.
  • an aqueous solution e.g. 50 mM potassium phosphate solution at pH 7, or distilled H 2 0, to concentrations of 25-50 ⁇ .
  • Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J- 710) using standard measurement parameters (e.g., temperature, 20°C; wavelength, 190- 260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm).
  • the a-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g., [ ⁇ ]222obs) by the reported value for a model helical decapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).
  • a peptidomimetic macrocycle of the invention comprising a secondary structure such as an a-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide.
  • peptidomimetic macrocycles of the invention exhibit Tm of > 60°C representing a highly stable structure in aqueous solutions.
  • Tm is determined by measuring the change in ellipticity over a temperature range (e.g,.
  • spectropolarimeter e.g., Jasco J-710
  • standard parameters e.g. wavelength 222nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: l°C/min; path length, 0.1 cm.
  • the peptidomimetic macrocycles of the present invention may be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide.
  • the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm.
  • the peptidomimetic macrocycle and peptidomimetic precursor (5 meg) are incubated with trypsin agarose (Pierce) (S/E -125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC -based peak detection at 280 nm.
  • Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more.
  • an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more.
  • assays may be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 meg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37°C for 0, 1, 2, 4, 8, and 24 hours.
  • the samples are extracted by transferring 100 ⁇ of sera to 2 ml centrifuge tubes followed by the addition of 10 of 50 % formic acid and 500 ⁇ acetonitrile and centrifugation at 14,000 RPM for 10 min at 4 ⁇ 2°C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N 2 ⁇ 10 psi, 37°C. The samples are
  • FPA fluorescence polarization assay
  • fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25- lOOOnM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity ismeasured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA). A peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding uncrosslinked polypeptide. In vitro Displacement Assays To Characterize Antagonists of Peptide-Protein
  • FPA fluorescence polarization assay
  • FITC-labeled peptides bound to a large protein emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules ⁇ e.g. FITC-labeled peptides that are free in solution).
  • a compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.
  • putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.
  • Antagonist binding activity ismeasured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B).
  • Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).
  • Any class of molecule such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.
  • an affinity-selection mass spectrometry assay is used, for example.
  • Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system- wide control experiment using 1 ⁇ peptidomimetic macrocycle plus 5 ⁇ target protein.
  • a 1 iL DMSO aliquot of a 40 ⁇ stock solution of peptidomimetic macrocycle is dissolved in 19 ⁇ _, of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl).
  • PBS Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl.
  • the resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min.
  • Each 8.0 ⁇ ⁇ experimental sample thus contains 40 pmol (1.5 ⁇ g) of protein at 5.0 ⁇ concentration in PBS plus 1 ⁇ peptidomimetic macrocycle and 2.5% DMSO.
  • Duplicate samples thus prepared for each concentration point are incubated for 60 min at room temperature, and then chilled to 4 °C prior to size-exclusion chromatography-LC-MS analysis of 5.0 injections.
  • Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding component by a rapid SEC step.
  • the SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column.
  • the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism.
  • the (M + 3H) ion of the peptidomimetic macrocycle is observed by ESI-MS at the expected m/z, confirming the detection of the protein-ligand complex.
  • Protein-ligand Kd titrations experiments are conducted as follows: 2 ⁇ DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, 0.098 mM) are prepared then dissolved in 38 of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To 4.0 ⁇ aliquots of the resulting supernatants is added 4.0 ⁇ ⁇ of 10 ⁇ hMDM2 in PBS.
  • Each 8.0 ⁇ ⁇ experimental sample thus contains 40 pmol (1.5 ⁇ g) of protein at 5.0 ⁇ concentration in PBS, varying concentrations (125, 62.5, 0.24 ⁇ ) of the titrant peptide, and 2.5% DMSO.
  • Duplicate samples thus prepared for each concentration point are incubated at room temperature for 30 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 ⁇ injections.
  • an affiinity selection mass spectrometry assay is performed, for example.
  • a mixture of ligands at 40 ⁇ per component is prepared by combining 2 ⁇ aliquots of 400 ⁇ stocks of each of the three compounds with 14 ⁇ of DMSO. Then, 1 ⁇ aliquots of this 40 ⁇ per component mixture are combined with 1 ⁇ DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, 0.078 mM). These 2 ⁇ samples are dissolved in 38 ⁇ , of PBS.
  • the resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min.
  • To 4.0 ⁇ ⁇ aliquots of the resulting supernatants is added 4.0 ⁇ ⁇ of 10 ⁇ hMDM2 protein in PBS.
  • Each 8.0 ⁇ ⁇ experimental sample thus contains 40 pmol (1.5 ⁇ g) of protein at 5.0 ⁇ concentration in PBS plus 0.5 ⁇ ligand, 2.5% DMSO, and varying concentrations (125, 62.5, 0.98 ⁇ ) of the titrant peptidomimetic macrocycle.
  • Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 ⁇ injections.
  • Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 ⁇ goat anti-FITC antibody for 2 hrs, rotating at 4°C followed by further 2 hrs incubation at 4°C with protein A/G Sepharose (50 ⁇ of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle.
  • increasing salt concentration e.g. 150, 300, 500 m
  • uncrosslinked macrocycle intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 ⁇ ) for 4 hrs in serum free media at 37°C, washed twice with media and incubated with trypsin (0.25%) for 10 min at 37°C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan ® HCS Reader.
  • the efficacy of certain peptidomimetic macrocycles is determined, for example, in cell- based HIV infectivity assays using a variety of viral strains derived from human clinical populations. Cell viability is monitored, for example, over 24-96 hrs of incubation with peptidomimetic macrocycles (1 uM to 10 ⁇ ) to identify those that prevent HIV infection at EC50 ⁇ 10 ⁇ .
  • peptidomimetic macrocycles (1 uM to 10 ⁇
  • the compounds are, for example,administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0', 5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 ⁇ , of fresh serum are then measured by LC-MS/MS as above.
  • Clinical Trials To determine the suitability of the peptidomimetic macrocycles of the invention for treatment of humans, clinical trials are performed. For example, patients diagnosed with HIV infection and in need of treatment are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle of the invention, while the control groups receive a placebo or a known anti- HIV drug.
  • the treatment safety and efficacy of the peptidomimetic macrocycles of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life.
  • the patient group treated with a peptidomimetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo.
  • the peptidomimetic macrocycles of the invention also include pharmaceutically
  • a "pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention.
  • Particularly favored pharmaceutically acceptable derivatives are those that increase the bioavailability of the compounds of the invention when administered to a mammal (e.g., by increasing absorption into the blood of an orally administered compound) or which increases delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.
  • Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility or active transport across the gastrointestinal mucosa.
  • the peptidomimetic macrocycles of the invention are modified by covalently or non-covalently joining appropriate functional groups to enhance selective biological properties.
  • modifications include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.
  • Suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.
  • Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl) 4 + salts.
  • pharmaceutically acceptable carriers include either solid or liquid carriers.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA.
  • the carrier is a finely divided solid, which is in a mixture with the finely
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl- cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
  • liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the pharmaceutical preparation is preferably in unit dosage form. In such form the
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • compositions of this invention comprise a combination of a peptidomimetic macrocycle and one or more additional therapeutic or prophylactic agents
  • both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
  • the additional agents are administered separately, as part of a multiple dose regimen, from the compounds of this invention.
  • those agents are part of a single dosage form, mixed together with the compounds of this invention in a single composition.
  • the amount of macrocycle that can be administered is not limited, and can range, for example, from about 2000 mg to about 1 mg, from about 1000 mg to about 1 mg, from about 500 mg to about 1 mg, or from about 100 mg to about 1 mg.
  • Example 1 Peptidomimetic macrocycles of the invention
  • Peptidomimetic macrocycles were designed by replacing two or more naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions.
  • Non-natural amino acids (4 equiv) were coupled with a 1 : 1 :2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA.
  • the N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.
  • Tables 3 and 4 show a list of peptidomimetic macrocycles of the invention prepared.
  • Nle represents norleucine
  • Aib represents 2-aminoisobutyric acid
  • Ac represents acetyl
  • Pr represents propionyl.
  • Amino acids represented as “$” are alpha-Me S5- pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond.
  • Amino acids represented as "$r5" are alpha-Me R5- pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond.
  • Amino acids represented as "$s8" are alpha-Me S8-octenyl- alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond.
  • Amino acids represented as "$r8” are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond.
  • “Ahx” represents an aminocyclohexyl linker.
  • the crosslinkers are linear all-carbon crosslinker comprising eight or eleven carbon atoms between the alpha carbons of each amino acid.
  • Amino acids represented as "$/” are alpha-Me S5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker.
  • Amino acids represented as "$/r5" are alpha-Me R5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker.
  • Amino acids represented as "$/s8” are alpha-Me S8-octenyl-alanine olefin amino acids that are not connected by any crosslinker.
  • Amino acids represented as "$/r8” are alpha-Me R8-octenyl-alanine olefin amino acids that are not connected by any crosslinker.
  • Amino acids represented as "Amw” are alpha-Me tryptophan amino acids.
  • Amino acids represented as “Ami” are alpha-Me leucine amino acids.
  • Amino acids represented as "2ff ' are 2-fluoro-phenylalanine amino acids.
  • Amino acids represented as "3ff ' are 3 -fluoro-phenyl alanine amino acids.
  • Amino acids represented as "St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated.
  • Amino acids represented as “St//” are amino acids comprising two pentenyl-alanine olefin side chains that are not crosslinked.
  • Amino acids represented as "%St” are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated via fully saturated hydrocarbon crosslinks.
  • Fresh human PBMCs, seronegative for HIV and HBV, are isolated from screened donors (Biological Specialty Corporation, Colmar, PA). Cells are pelleted/washed 2-3 times by low speed centrifugation and re-suspension in PBS to remove contaminating platelets.
  • the Leukophoresed blood is then diluted 1 : 1 with Dulbecco's Phosphate Buffered Saline (DPBS) and layered over 14 mL of Lymphocyte Separation Medium (LSM; Cellgro® by Mediatech, Inc.; density 1.078+/-0.002 g/ml; Cat.# 85-072-CL) in a 50 mL centrifuge tube and then centrifuged for 30 minutes at 600 X g. Banded PBMCs are gently aspirated from the resulting interface and subsequently washed 2X with PBS by low speed centrifugation.
  • DPBS Dulbecco's Phosphate Buffered Saline
  • cells are enumerated by trypan blue exclusion and re-suspended at 1 x 107 cells/mL in RPMI 1640 supplemented with 15 % Fetal Bovine Serum (FBS), and 2 mM L-glutamine, 4 ⁇ g/mL Phytohemagglutinin (PHA, Sigma). The cells are allowed to incubate for 48-72 hours at 37°C.
  • FBS Fetal Bovine Serum
  • PHA Phytohemagglutinin
  • PBMCs are centrifuged and re-suspended in RPMI 1640 with 15% FBS, 2 mM L- glutamine, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 20 U/mL recombinant human IL-2 (R&D Systems, Inc).
  • IL-2 is included in the culture medium to maintain the cell division initiated by the PHA mitogenic stimulation.
  • PBMCs are maintained in this medium at a concentration of 1-2 x 106 cells/mL with biweekly medium changes until used in the assay protocol. Cells are kept in culture for a maximum of two weeks before being deemed too old for use in assays and discarded. MDMs are depleted from the culture as the result of adherence to the tissue culture flask.
  • PHA stimulated cells from at least two normal donors are pooled (mixed together), diluted in fresh medium to a final concentration of 1 x 106 cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 ⁇ (5 x 104 cells/well) in a standard format. Pooling (mixing) of mononuclear cells from more than one donor is used to minimize the variability observed between individual donors, which results from quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 of primary lymphocyte populations.
  • Each plate contains virus/cell control wells (cells plus virus), experimental wells (drug plus cells plus virus) and compound control wells (drug plus media without cells, necessary for MTS monitoring of cytotoxicity).
  • virus/cell control wells cells plus virus
  • experimental wells drug plus cells plus virus
  • compound control wells drug plus media without cells, necessary for MTS monitoring of cytotoxicity.
  • Test drug dilutions are prepared at a 2X concentration in microtiter tubes and 100 of each concentration (nine total concentrations) are placed in appropriate wells using the standard format. 50 ⁇ ⁇ of a predetermined dilution of virus stock is placed in each test well (final MOI ⁇ 0.1).
  • the PBMC cultures are maintained for seven days following infection at 37°C, 5% C02. After this period, cell-free supernatant samples are collected for analysis of reverse transcriptase activity and/or p24 antigen content. Following removal of supernatant samples, compound cytotoxicity is measured by addition of MTS to the plates for determination of cell viability. Wells are also examined microscopically and any abnormalities are noted.
  • RT reverse transcriptase
  • Poly rA:oligo dT template :primer (Pharmacia) is prepared as a stock solution by combining 150 poly rA (20 mg/mL) with 0.5 mL oligo dT (20 units/mL) and 5.35 mL sterile dH20 followed by aliquoting (1.0 mL) and storage at -20°C.
  • the RT reaction buffer is prepared fresh on a daily basis and consists of 125 1.0 M EGTA, 125 ⁇ , dH20, 125 ⁇ 20% Triton X100, 50 ⁇ 1.0 M Tris (pH 7.4), 50 ⁇ 1.0 M DTT, and 40 ⁇ 1.0 M MgC12.
  • the final reaction mixture is prepared by combining 1 part 3H-TTP, 4 parts dH20, 2.5 parts poly rA:oligo dT stock and 2.5 parts reaction buffer. Ten microliters of this reaction mixture is placed in a round bottom microtiter plate and 15 ⁇ of virus containing supernatant is added and mixed. The plate is incubated at 37°C for 60 minutes. Following incubation, the reaction volume is spotted onto DE81 filter-mats (Wallac), washed 5 times for 5 minutes each in a 5% sodium phosphate buffer or 2X SSC (Life Technologies). Next they are washed 2 times for 1 minute each in distilled water, 2 times for 1 minute each in 70% ethanol, and then dried. Incorporated radioactivity (counts per minute, CPM) is quantified using standard liquid scintillation techniques.
  • Example 4 Anti-HIV Efficacy Evaluation in Fresh Human MDMs
  • Peripheral blood monocytes are isolated from screened donors, seronegative for HIV and HBV. Cells are pelleted/washed 2-3 times by low speed centrifugation and re-suspension in PBS to remove contaminating platelets. The Leukophoresed blood is then diluted 1 : 1 with Dulbecco's phosphate buffered saline (PBS) and layered over 14 mL of PBS.
  • PBS Dulbecco's phosphate buffered saline
  • Lymphocyte Separation Medium (LSM; cellgro® by Mediatech, Inc.; density 1.078+/- 0.002 g/ml; Cat.# 85-072-CL) in a 50 mL centrifuge tube and then centrifuged for 30 minutes at 600 X g. Banded PBMCs are gently aspirated from the interface and subsequently washed 2X with PBS by low speed centrifugation. The cells are diluted to 4 x 106 cells per mL in DMEM without phenol red supplemented with 10% heat inactivated human pooled AB serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin.
  • LSM Lymphocyte Separation Medium
  • Monocytes-derived-macrophages are allowed to adhere to the interior 60 wells (100 ⁇ ) of a 96 well flat bottomed plate for 2 to 18 hours at 37°C, 5% C02.
  • the exterior wells are filled with 200 of sterile DPBS to serve as a humidity barrier. Following adherence, the cultures are washed with sterile DPBS to remove non-adherent cells (lymphocytes and contaminating RBCs).
  • 200 L of RPMI 1640 supplemented with 15 % FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin is subsequently added to the wells.
  • the plates are incubated at 37°C in a humidified incubator with 5% C02. Culture medium is replaced once per week until use. Culture plates are used for anti-HIV evaluations between days 6 and 14 of incubation following initial isolation of the cells.
  • the MDM cultures are washed 3 times to remove any non-adherent cells and serially diluted test compounds are added followed by the addition of a pre-titered amount of HIV. Cultures are washed a final time by media removal 24 hours post infection, fresh compound added and the cultures continued for an additional six days.
  • the assays are performed using a standardized microtiter plate format. Each plate contains virus/cell control wells (cells plus virus), experimental wells (drug plus cells plus virus) and compound control wells (drug plus media without cells, necessary for MTS monitoring of cytotoxicity). In this in vitro assay, macrophage viability remains high throughout the duration of the incubation period.
  • virus replication is measured by collecting cell-free supernatant samples, which are analyzed for HIV-1 p24 antigen content using a commercially available p24 ELISA assay (e.g., PerkinElmer).
  • p24 ELISA assay e.g., PerkinElmer
  • compound cytotoxicity is measured by addition of MTS to the plates for determination of cell viability.
  • Wells are also examined microscopically and any abnormalities noted.
  • P24 antigen ELISA kits are purchased from PerkinElmer or other similar vendors. The assay is performed according to the manufacturer's instructions. Control curves are generated in each assay to accurately quantify the amount of p24 antigen in each sample. Data are obtained by spectrophotometric analysis at 490/670 nm using a Molecular Devices SpectraMaxPlus plate reader. Final concentrations are calculated from the optical density values using the Molecular Devices SOFTmax Pro software package.

Abstract

Provided are novel peptidomimetic macrocycles and methods of using the macrocycles for the treatment of disease.

Description

PEPTIDOMIMETIC MACROCYCLES
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Application Serial No. 61/548,695, filed October 18, 2012.
BACKGROUND OF THE INVENTION
[0002] Entry of HIV- 1 into cells is an ordered, multi-step process leading to fusion of viral and cell membranes. The first step in entry is binding of the viral envelope to CD4 on the surface of macrophages or T-helper lymphocytes. Binding to CD4 is mediated by gpl20, the surface subunit of the envelope. In its native form, the envelope glycoprotein is a heterotrimer of three gpl20 and three HIV HIV gp41 molecules. Conformational changes triggered by CD4 binding, and other structural alterations, allows the N-terminus of HIV gp41 to insert into the target cell membrane, resulting in fusion and viral infection. Enfuvirtide (FUZEON®, T-20), the first HIV entry inhibitor, is a 36-mer synthetic oligopeptide derived from the HR2 region of the HIV-1 envelope HIV gp41 subunit. Binding of enfuvirtide to HIV gp41 prevents the formation of a six -helix bundle between the HR1 and HR2 helices of three HIV gp41 proteins. This HIV HIV gp41 six- helix bundle is required for proper HIV fusion with the target cell, and antagonism of the fusion complex formation blocks virus entry and prevents propagation of wild-type and resistant strains. Because the drug is an oligopeptide, it is administered subcutaneous ly by injection. While enfuvirtide is effective and has minimal systemic toxicity, its short half-life and frequent painful injection site reactions (with twice daily dosing) have limited its use to late line therapy in treatment experienced patients. While new agents have lowered resistance rates in the near-term, resistance continues to be recognized as an increasing problem over next 3-7 years with second generation HIV combinations, and thus there is a clinical need for improved HIV fusion inhibitors with less frequent dosing schedules and reduced injection site reactions.
SUMMARY OF THE INVENTION
[0003] The Summary of the Invention, being illustrative and selective, does not limit the scope of any claim, does not provide the entire scope of inventive embodiments disclosed or contemplated herein, and should not be construed as limiting or constraining the scope of this disclosure or any claimed inventive embodiment. [0004] The present invention provides HIV gp41 -derived peptidomimetic macrocycles that are designed to possess improved pharmaceutical properties relative to enfuvirtide. These improved properties include enhanced chemical and in vivo stability, increased potency and reduced immunogenicity {i.e., fewer or less severe injection site reactions). These peptidomimetic macrocycles are useful for the prevention and treatment of HIV infection and resulting immunedeficiencies {i.e., AIDS).
[0005] Described below are stably cross-linked peptides related to a portion of the HIV HIV gp41 protein. These cross-linked peptides contain at least two modified amino acids that together form an intramolecular cross-link that can help to stabilize the alpha-helical secondary structures of a portion of HIV HIV gp41 that is thought to be important for assembly of the HIV gp41 six-helix bundle fusion complex. Accordingly, a cross-linked polypeptide described herein can have improved biological activity relative to a corresponding polypeptide that is not cross-linked. The HIV HIV gp41 peptidomimetic macrocycles are thought to interfere with binding of HIV gp41 HR1 and HR2 helices to each other to prevent assembly of the active six-helix bundle fusion complex, thereby inhibiting entry of the HIV virus. The HIV HIV gp41 peptidomimetic macrocycles described herein can be used therapeutically, for example, to prevent or to treat HIV infection and resulting immunedeficiencies {i.e., AIDS).
[0006] Provided herein is a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1, 2, 3, and 4, wherein the
peptidomimetic macrocycle has an IC50 of about 10000 nM or less against a target and in cells selected from the group consisting of: target: HIV-1 IIIB, cells: CEM-SS; target: HIV-1NL4-3, cells: MT-4; and target: HIV-1MDR769, cells: hPBMC, wherein the peptidomimetic macrocycle is not a compound having the sequence:
(SP-13)YT$LI H$LIEESQNQQEKNEQELLEL$KW A $ L W NWF,
(SP-33) MeTWSEWD $EINNYTSLIHSL IEESQN$QEK$EQE L L E, and
(SP-41)NleTW$EWD$EINNYT$L IH$LI EESQNQ$E KN $ Q ELLE.
[0007] Also provided herein is a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to HIV gp41626-663, further comprising at least two independent crosslinkers, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid, wherein the first amino acid and the second amino acid are selected from the group consisting of amino acids: 629 and 633; 630 and 634; 632 and 636; and 633 and 637; wherein the third amino acid and the fourth amino acid are selected from the group consisting of amino acids: 650 and 654; 651 and 655; 652 and 656; 653 and 657; 654 and 658; 655 and 659; 656 and 660; and 657 and 771;with the proviso that when the first crosslinker connects amino acids pairs 629 and 633 and the second crosslinker connects amino acid pair 653 and 657, there is an additional crosslinker that connects amino acids pairs 641 and 645 or 642 and 646.
[0008] Further provided herein is a peptidomimetic macrocycle comprising an amino acid
sequence which is at least about 60% identical to HIV gp41 638-673, further having one crosslinker, wherein the crosslinker connects a first amino acid to a second amino acid, wherein the first amino acid and the second amino acid are 652 and 659.
[0009] Additionally provided herein is a peptidomimetic macrocycle which is at least about 60% identical to HIV gp41 638-673, further comprising at at least two independent crosslinkers, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid, wherein the amino acid pairs are selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671; wherein when the first crosslinker connects amino acid pair 640 and 644, the second crosslinker does not connect the amino acid pair 664 and 668 or 666 and 670; and
wherein when the first crosslinker connects amino acid pair 653 and 657, the second crosslinker does not connect amino acid pair 666 and 670.
[0010] Further provided herein are a method of preventing or treating HIV infection and
associated immunodeficiencies (i.e., AIDS) in a subject by administering an antagonist of the six -helix bundle HIV HIV gp41 fusion complex, wherein the antagonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week.
[0011] Also provided herein is a method of preventing or treating HIV infection and associated immunodeficiencies (i.e., AIDS) in a subject by administering an antagonist which can be a peptidomimetic macrocycle herein of the six -helix bundle HIV HIV gp41 fusion complex, wherein the frequency or intensity of the site of injection reaction is reduced relative to that of enfuvirtide.
[0012] Additionally, provided herein is a method of preventing or treating HIV infection and associated immunodeficiencies (i.e., AIDS) in a subject comprising administering to the subject a peptidomimetic macrocycle of the invention.
[0013] Also provided is a method of inhibiting HIV viral entry in a subject comprising
administering to the subject a peptidomimetic macrocycle of the invention, or a method of antagonizing the formation of the six -helix bundle HIV HIV gp41 fusion complex in a subject comprising administering to the subject such a peptidomimetic macrocycle herein.
INCORPORATION BY REFERENCE
[0014] All publications, patents, and patent applications referenced herein are incorporated by reference in their entireties. In the event of a conflict between a term herein and a term incorporated by reference, the term herein controls.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The novel features of the invention are set forth with particularity in the appended claims.
A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0016] FIGURE 1 shows improved stabilities to trypsin proteolysis of the peptidomimetic
macrocycles of the invention.
[0017] FIGURE 2 shows in vitro anti-viral activities of the peptidomimetic macrocycles of the invention.
[0018] FIGURE 3 shows in vitro anti-viral activities of the peptidomimetic macrocycles of the invention
DETAILED DESCRIPTION OF THE INVENTION
[0019] Herein, unless otherwise indicated, the article "a" means one or more unless explicitly otherwise provided for.
[0020] Herein, unless otherwise indicated, terms such as "contain," "containing," "include,"
"including," and the like mean "comprising."
[0021] Herein, unless otherwise indicated, the term "or" can be conjunctive or disjunctive. [0022] Herein, unless otherwise indicated, any embodiment can be combined with any other embodiment.
[0023] Herein, unless otherwise indicated, some inventive embodiments contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every subrange and value within the range is present as if explicitly written out.
[0024] As used herein, the term "macrocycle" refers to a molecule having a chemical structure including a ring or cycle formed by at least 9 covalently bonded atoms.
[0025] As used herein, the term "peptidomimetic macrocycle" or "crosslinked polypeptide" refers to a compound comprising a plurality of amino acid residues joined by a plurality of peptide bonds and at least one macrocycle-forming linker which forms a macrocycle between a first naturally-occurring or non-naturally-occurring amino acid residue (or analog) and a second naturally-occurring or non-naturally-occurring amino acid residue (or analog) within the same molecule. Peptidomimetic macrocycle include embodiments where the macrocycle-forming linker connects the a carbon of the first amino acid residue (or analog) to the a carbon of the second amino acid residue (or analog). The peptidomimetic macrocycles optionally include one or more non-peptide bonds between one or more amino acid residues and/or amino acid analog residues, and optionally include one or more non-naturally-occurring amino acid residues or amino acid analog residues in addition to any which form the macrocycle. A "corresponding uncrosslinked polypeptide" when referred to in the context of a peptidomimetic macrocycle is understood to relate to a polypeptide of the same length as the macrocycle and comprising the equivalent natural amino acids of the wild-type sequence corresponding to the macrocycle. Peptidomimetic macrocycles include, for example: macrocycles having one crosslinker (e.g., stapled peptidomimetic macrocycles); macrocycles having two or more crosslinkers that are not in tandem (e.g., multiply stapled peptidomimetic macrocycles); macrocycles having two crosslinkers that are in tandem (e.g. , stitched peptidomimetic macrocycles); and macrocycles that are stitched and further stapled with at least one additional crosslinker.
[0026] As used herein, the term "stability" refers to the maintenance of a defined secondary structure in solution by a peptidomimetic macrocycle of the invention as measured by circular dichroism, NMR or another biophysical measure, or resistance to proteolytic degradation in vitro or in vivo. Non-limiting examples of secondary structures contemplated in this invention are a-helices, β-turns, and β-pleated sheets. [0027] As used herein, the term "helical stability" refers to the maintenance of a helical structure by a peptidomimetic macrocycle of the invention as measured by circular dichroism or NMR. For example, in some embodiments, the peptidomimetic macrocycles of the invention exhibit at least a 1.25, 1.5, 1.75 or 2-fold increase in a-helicity as determined by circular dichroism compared to a corresponding uncrosslinked macrocycle.
[0028] The term "amino acid" refers to a molecule containing both an amino group and a
carboxyl group. Suitable amino acids include, without limitation, both the D-and L- isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes. The term amino acid, as used herein, includes without limitation, a-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.
[0029] The term "a-amino acid" refers to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the a-carbon.
[0030] The term "β-amino acid" refers to a molecule containing both an amino group and a carboxyl group in a β configuration.
[0031] The term "naturally occurring amino acid" refers to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
[0032] The term "amino acid analog" refers to a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, without limitation, β-amino acids and amino acids where the amino or carboxy group is substituted by a similarly reactive group (e.g. , substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).
[0033] The term "non-natural amino acid" refers to an amino acid which is not one of the the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
[0034] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially abolishing its essential biological or biochemical activity (e.g., receptor binding or activation). An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity. [0035] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predicted nonessential amino acid residue in a polypeptide, for example, is preferably replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine).
[0036] The term "member" as used herein in conjunction with macrocycles or macrocycle- forming linkers refers to the atoms that form or can form the macrocycle, and excludes substituent or side chain atoms. By analogy, cyclodecane, 1 ,2-difluoro-decane and 1,3- dimethyl cyclodecane are all considered ten-membered macrocycles as the hydrogen or fluoro substituents or methyl side chains do not participate in forming the macrocycle.
Figure imgf000008_0001
[0037] The symbol " when used as part of a molecular structure refers to a single bond or a trans or cis double bond.
[0038] The term "amino acid side chain" refers to a moiety attached to the a-carbon in an amino acid. For example, the amino acid side chain for alanine is methyl, the amino acid side chain for phenylalanine is phenylmethyl, the amino acid side chain for cysteine is thiomethyl, the amino acid side chain for aspartate is carboxymethyl, the amino acid side chain for tyrosine is 4-hydroxyphenylmethyl, etc. Other non-naturally occurring amino acid side chains are also included, for example, those that occur in nature (e.g., an amino acid metabolite) or those that are made synthetically (e.g., an α,α di-substituted amino acid).
[0039] The term "α,α di-substituted amino" acid refers to a molecule or moiety containing both an amino group and a carboxyl group bound to a carbon (the a-carbon) that is attached to two natural or non-natural amino acid side chains.
[0040] The term "polypeptide" encompasses two or more naturally or non-naturally-occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptides as described herein include full length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally-occurring proteins or synthetic polypeptide fragments). [0041] The term "macrocyclization reagent" or "macrocycle-forming reagent" as used herein refers to any reagent which may be used to prepare a peptidomimetic macrocycle of the invention by mediating the reaction between two reactive groups. Reactive groups may be, for example, an azide and alkyne, in which case macrocyclization reagents include, without limitation, Cu reagents such as reagents which provide a reactive Cu(I) species, such as CuBr, Cul or CuOTf, as well as Cu(II) salts such as Cu(C02CH3)2, CuS04, and CuCl2 that can be converted in situ to an active Cu(I) reagent by the addition of a reducing agent such as ascorbic acid or sodium ascorbate. Macrocyclization reagents may additionally include, for example, Ru reagents known in the art such as Cp*RuCl(PPh3)2, [Cp*RuCl]4 or other Ru reagents which may provide a reactive Ru(II) species. In other cases, the reactive groups are terminal olefins. In such embodiments, the
macrocyclization reagents or macrocycle-forming reagents are metathesis catalysts including, but not limited to, stabilized, late transition metal carbene complex catalysts such as Group VIII transition metal carbene catalysts. For example, such catalysts are Ru and Os metal centers having a +2 oxidation state, an electron count of 16 and
pentacoordinated. Additional catalysts are disclosed in Grubbs et al, "Ring Closing Metathesis and Related Processes in Organic Synthesis" Acc. Chem. Res. 1995, 28, 446- 452, and U.S. Pat. No. 5,811,515. In yet other cases, the reactive groups are thiol groups. In such embodiments, the macrocyclization reagent is, for example, a linker
functionalized with two thiol-reactive groups such as halogen groups.
[0042] The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine or a radical thereof.
[0043] The term "alkyl" refers to a hydrocarbon chain that is a straight chain or branched chain, containing the indicated number of carbon atoms. For example, Ci-Cio indicates that the group has from 1 to 10 (inclusive) carbon atoms in it. In the absence of any numerical designation, "alkyl" is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it. Alkyl groups can have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Alkyl groups include, for example, methyl, ethyl, n-propyl, iso-propyl, and butyl groups.
[0044] The term "alkylene" refers to a divalent alkyl (i.e., -R-).
[0045] The term "alkenyl" refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon double bonds. The alkenyl moiety contains the indicated number of carbon atoms. For example, C2-Cio indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term "lower alkenyl" refers to a C2-C6 alkenyl chain. In the absence of any numerical designation, "alkenyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
[0046] The term "alkynyl" refers to a hydrocarbon chain that is a straight chain or branched chain having one or more carbon-carbon triple bonds. The alkynyl moiety contains the indicated number of carbon atoms. For example, C2-C10 indicates that the group has from 2 to 10 (inclusive) carbon atoms in it. The term "lower alkynyl" refers to a C2-C6 alkynyl chain. In the absence of any numerical designation, "alkynyl" is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.
[0047] The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring are substituted by a substituent.
Examples of aryl groups include phenyl, naphthyl and the like.
[0048] "Arylalkyl" refers to an aryl group substituted with an aryl. For example, one of the aryl group's hydrogen atoms has been replaced with a C1-C5 alkyl group, as defined above. Representative examples of an arylalkyl group include, but are not limited to, 2- methylphenyl, 3-methylphenyl, 4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4- ethylphenyl, 2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl, 3- butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl, 4-pentylphenyl, 2- isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl, 2-isobutylphenyl, 3- isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl, 3-sec-butylphenyl, 4-sec- butylphenyl, 2-t-butylphenyl, 3-t-butylphenyl and 4-t-butylphenyl. The term "arylalkoxy" refers to an alkoxy substituted with aryl.
[0049] "Arylamido" refers to an aryl group, as defined above, wherein one of the aryl group's hydrogen atoms has been replaced with one or more -C(0)NH2 groups. Representative examples of an arylamido group include 2-C(0)NH2 -phenyl, 3-C(0)NH2-phenyl, 4- C(0)NH2-phenyl, 2-C(0)NH2-pyridyl, 3-C(0)NH2-pyridyl, and 4-C(0)NH2-pyridyl,
[0050] "Alkylheterocycle" refers to a C1-C5 alkyl group, as defined above, wherein one of the
C1-C5 alkyl group's hydrogen atoms has been replaced with a heterocycle. Representative examples of an alkylheterocycle group include, but are not limited to, -CH2CH2- morpholine, -CH2CH2-piperidine, -CH2CH2CH2-morpholine, and -CH2CH2CH2- imidazole.
[0051] "Alkylamido" refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a -C(0)NH2 group. Representative examples of an alkylamido group include, but are not limited to, -CH2-C(0)NH2, - CH2CH2-C(0)NH2, -CH2CH2CH2C(0)NH2, -CH2CH2CH2CH2C(0)NH2, - CH2CH2CH2CH2CH2C(0)NH2, -CH2CH(C(0)NH2)CH3, -CH2CH(C(0)NH2)CH2CH3, - CH(C(0)NH2)CH2CH3, -C(CH3)2CH2C(0)NH2, -CH2-CH2-NH-C(0)-CH3, -CH2-CH2- NH-C(0)-CH3-CH3, and -CH2-CH2-NH-C(0)-CH=CH2.
[0052] "Alkanol" refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a hydroxyl group. Representative examples of an alkanol group include, but are not limited to, -CH2OH, -CH2CH2OH, - CH2CH2CH2OH, -CH2CH2CH2CH2OH, -CH2CH2CH2 CH2CH2OH, -CH2CH(OH)CH3, - CH2CH(OH)CH2CH3, -CH(OH)CH3 and -C(CH3)2CH2OH.
[0053] "Alkylcarboxy" refers to a C1-C5 alkyl group, as defined above, wherein one of the C1-C5 alkyl group's hydrogen atoms has been replaced with a --COOH group. Representative examples of an alkylcarboxy group include, but are not limited to, -CH2COOH, - CH2CH2COOH, -CH2CH2CH2COOH, -CH2CH2CH2CH2COOH, -CH2CH(COOH)CH3, - CH2CH2CH2CH2CH2COOH, -CH2CH(COOH)CH2CH3, -CH(COOH)CH2CH3 and - C(CH3)2CH2COOH.
[0054] The term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally is optionally substituted. Some cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclop entenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
[0055] The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1 -3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2, 3, or 4 atoms of each ring are substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.
[0056] The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.
[0057] The term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12
membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2 or 3 atoms of each ring are substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
[0058] The term "substituent" refers to a group replacing a second atom or group such as a
hydrogen atom on any molecule, compound or moiety. Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.
[0059] In some embodiments, the compounds of this invention contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are included in the present invention unless expressly provided otherwise. In some embodiments, the compounds of this invention are also represented in multiple tautomeric forms, in such instances, the invention includes all tautomeric forms of the compounds described herein (e.g. , if alkylation of a ring system results in alkylation at multiple sites, the invention includes all such reaction products). All such isomeric forms of such compounds are included in the present invention unless expressly provided otherwise. All crystal forms of the compounds described herein are included in the present invention unless expressly provided otherwise.
[0060] As used herein, the terms "increase" and "decrease" mean, respectively, to cause a
statistically significantly (i.e., p < 0.1) increase or decrease of at least 5%.
[0061] The term "on average" represents the mean value derived from performing at least three independent replicates for each data point.
[0062] The term "biological activity" encompasses structural and functional properties of a macrocycle of the invention. Biological activity is, for example, structural stability, alpha-helicity, affinity for a target, resistance to proteolytic degradation, cell
penetrability, intracellular stability, in vivo stability, or any combination thereof.
[0063] In some embodiments, the peptide sequences are derived from the HIV HIV gp41
protein.
[0064] A non-limiting exemplary list of suitable HIV HIV gp41 peptides for use in the present invention is given in Table 1 and Table 2 below (X residues form cross-links to one other X residue, Z residues form cross-links to one other Z residue, and XX residues form cross-links with two other X residues):
TABLE 1 AC-YT-X-LIH-X-LIEESQN-Z-QEK-Z-EQELLELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQN-Z-QEKNEQ-Z-LLELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQ-Z-EKN-Z-QELLELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQ-Z-EKNEQ-Z-LLELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQ-Z-KNE-Z-ELLELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQ-Z-KNEQEL-Z-ELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQE-Z-NEQ-Z-LLELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQE-Z-NEQELL-Z-LDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEK-Z-EQE-Z-LELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEK-Z-EQELLE-Z-DKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEK-Z-EQL-Z-ELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEK-Z-EQLLEL-Z-KWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKN-Z-QLL-Z-LDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKN-Z-QLLELD-Z-WASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNE-Z-LLE-Z-DKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNE-Z-LLELDK-Z-ASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQ-Z-LEL-Z-KWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQ-Z-LELDKW-Z-SLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQL-Z-ELD-Z-WASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQL-Z-ELDKWASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLL-Z-LDK-Z-ASLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLL-Z-LDKWA-Z-LWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLE-Z-DKW-Z-SLWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLE-Z-DKWAS-Z-WNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLEL-Z-KWA-Z-LWNWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLEL-Z-KWASL-Z-NWF-NH2 Ac-YT-X- LIH-X-LIEESQNQQEEKNEQLLE L D-Z-WAS-Z-W N W F- N H 2 Ac-YT-X- LIH-X-LIEESQNQQEEKNEQLLE L D-Z-WAS LW-Z-W F-N H 2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLELDK-Z-ASL-Z-NWF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLELDK-Z-ASLWN-Z-F-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLELDKW-Z-SLW-Z-WF-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLELDKW-Z-SLWNW-Z-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLELDKWA-Z-LWN-Z-F-NH2 AC-YT-X-LIH-X-LIEESQNQQEEKNEQLLELDKWAS-Z-WNW-Z-NH2 AC-YT-X-LIHSLI-X-ESQN-Z-QEK-Z-EQELLELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQN-Z-QEKNEQ-Z-LLELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQ-Z-EKN-Z-QELLELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQ-Z-EKNEQ-Z-LLELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQ-Z-KNE-Z-ELLELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQ-Z-KNEQEL-Z-ELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQE-Z-NEQ-Z-LLELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQE-Z-NEQELL-Z-LDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEK-Z-EQE-Z-LELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEK-Z-EQELLE-Z-DKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEK-Z-EQL-Z-ELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEK-Z-EQLLEL-Z-KWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKN-Z-QLL-Z-LDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKN-Z-QLLELD-Z-WASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNE-Z-LLE-Z-DKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNE-Z-LLELDK-Z-ASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQ-Z-LEL-Z-KWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQ-Z-LELDKW-Z-SLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQL-Z-ELD-Z-WASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQL-Z-ELDKWASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLL-Z-LDK-Z-ASLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLL-Z-LDKWA-Z-LWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLE-Z-DKW-Z-SLWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLE-Z-DKWAS-Z-WNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLEL-Z-KWA-Z-LWNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLEL-Z-KWASL-Z-NWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELD-Z-WAS-Z-WNWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELD-Z-WASLW-Z-WF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELDK-Z-ASL-Z-NWF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELDK-Z-ASLWN-Z-F-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELDKW-Z-SLW-Z-WF-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELDKW-Z-SLWNW-Z-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELDKWA-Z-LWN-Z-F-NH2 AC-YT-X-LIHSLI-X-ESQNQQEEKNEQLLELDKWAS-Z-WNW-Z-NH2 AC-YTS-X-IHS-X-IEESQN-Z-QEK-Z-EQELLELDKWASLWNWF-NH2 AC-YTS-X-IHS-X-IEESQN-Z-QEKNEQ-Z-LLELDKWASLWNWF-NH2 AC-YTS-X-IHS-X-IEESQNQ-Z-EKN-Z-QELLELDKWASLWNWF-NH2 AC-YTS-X-IHS-X-IEESQNQ-Z-EKNEQ-Z-LLELDKWASLWNWF-NH2 AC-YTS-X-IHS-X-IEESQNQQ-Z-KNE-Z-ELLELDKWASLWNWF-NH2 AC-YTS-X-IHS-X-IEESQNQQ-Z-KNEQEL-Z-ELDKWASLWNWF-NH2 AC-YTS-X-IHS-X-IEESQNQQE-Z-NEQ-Z-LLELDKWASLWNWF-NH2 Ac-YTS-X- IHS-X-IEESQNQQE-Z-NEQELL-Z-LD KWAS LW N W F- N H 2 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AC-YTSLIHSL-X-EESQNQ-X-EEKN-Z-QLLELD-Z-WASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNE-Z-LLE-Z-DKWASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNE-Z-LLELDK-Z-ASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQ-Z-LEL-Z-KWASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQ-Z-LELDKW-Z-SLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQL-Z-ELD-Z-WASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQL-Z-ELDKWASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLL-Z-LDK-Z-ASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLL-Z-LDKWA-Z-LWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLE-Z-DKW-Z-SLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLE-Z-DKWAS-Z-WNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLEL-Z-KWA-Z-LWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLEL-Z-KWASL-Z-NWF-NH2 Ac-YTS LIHSL-X-EESQNQ-X-EEKNEQLLE L D-Z-WAS-Z-W N W F- N H 2 Ac-YTS LIHSL-X-EESQNQ-X-EEKNEQLLE L D-Z-WAS LW-Z-W F-N H 2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLELDK-Z-ASL-Z-NWF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLELDK-Z-ASLWN-Z-F-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLELDKW-Z-SLW-Z-WF-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLELDKW-Z-SLWNW-Z-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLELDKWA-Z-LWN-Z-F-NH2 AC-YTSLIHSL-X-EESQNQ-X-EEKNEQLLELDKWAS-Z-WNW-Z-NH2 AC-YT-X-LIH-XX-LIE-X-SQNQQEKKEQELLELDKWASLWNWF-NH2 AC-YTS-X-IHS-XX-IEE-X-QNQQEKNEQELLELDKWASLWNWF-NH2 AC-YTSL-X-HSL-XX-EES-X-NQQEKNEQELLELDKWASLWNWF-NH2 AC-YTSLI-X-SLI-XX-ESQ-X-QQEKNEQELLELDKWASLWNWF-NH2 AC-YTSLIH-X-LIE-XX-SQN-X-QEKNEQELLELDKWASLWNWF-NH2 AC-YTSLIHS-X-IEE-XX-QNQ-X-EKNEQELLELDKWASLWNWF-NH2 AC-YTSLIHSL-X-EES-XX-NQQ-X-KNEQELLELDKWASLWNWF-NH2 AC-YTSLIHSLI-X-ESQ-XX-QQE-X-NEQELLELDKWASLWNWF-NH2 AC-YTSLIHSLIE-X-SQN-XX-QEK-X-EQELLELDKWASLWNWF-NH2 AC-YTSLIHSLIEE-X-QNQ-XX-EKN-X-QELLELDKWASLWNWF-NH2 AC-YTSLIHSLIEES-X-NQQ-XX-KNE-X-ELLELDKWASLWNWF-NH2 AC-YTSLIHSLIEESQ-X-QQE-XX-NEQ-X-LLELDKWASLWNWF-NH2 AC-YTSLIHSLIEESQN-X-QEK-XX-EQE-X-LELDKWASLWNWF-NH2 AC-YTSLIHSLIEESQNQ-X-EKN-XX-QEL-X-ELDKWASLWNWF-NH2 AC-YTSLIHSLIEESQNQQ-X-KNE-XX-ELL-X-LDKWASLWNWF-NH2 AC-YTSLIHSLIEESQNQQE-X-NEQ-XX-LLE-X-DKWASLWNWF-NH2 AC-YTSLIHSLIEESQNQQEK-X-EQE-XX-LEL-X-KWASLWNWF-NH2 AC-YTSLIHSLIEESQNQQEKN-X-QEL-XX-ELD-X-WASLWNWF-NH2 AC-YTSLIHSLIEESQNQQEKNE-X-ELL-XX-LDK-X-ASLWNWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQ-X-LLE-XX-DKW-X-SLWNWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQE-X-LEL-XX-KWA-X-LWNWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQEL-X-ELD-XX-WAS-X-WNWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQELL-X-LDK-XX-ASL-X-NWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQELLE-X-DKW-XX-SLW-X-WF-NH2 AC-YTSLIHSLIEESQNQQEKNEQELLEL-X-KWA-XX-LWN-X-F-NH2 AC-YTSLIHSLIEESQNQQEKNEQELLELD-X-WAS-XX-WNW-X-NH2 AC-YT-X-LIH-XX-LIEESQ-X-QQEKNEQELLELDKWASLWNWF-NH2 AC-YTS-X-IHS-XX-IEESQN-X-QEKNEQELLELDKWASLWNWF-NH2 AC-YTSL-X-HSL-XX-EESQNQ-X-EKNEQELLELDKWASLWNWF-NH2 AC-YTSLI-X-SLI-XX-ESQNQQ-X-KNEQELLELDKWASLWNWF-NH2 AC-YTSLIH-X-LIE-XX-SQNQQE-X-NEQELLELDKWASLWNWF-NH2 AC-YTSLIHS-X-IEE-XX-QNQQEK-X-EQELLELDKWASLWNWF-NH2 AC-YTSLIHSL-X-EES-XX-NQQEKN-X-QELLELDKWASLWNWF-NH2 AC-YTSLIHSLI-X-ESQ-XX-QQEKNE-X-ELLELDKWASLWNWF-NH2 AC-YTSLIHSLIE-X-SQN-XX-QEKNEQ-X-LLELDKWASLWNWF-NH2 AC-YTSLIHSLIEE-X-QNQ-XX-EKNEQE-X-LELDKWASLWNWF-NH2 AC-YTSLIHSLIEES-X-NQQ-XX-KNEQEL-X-ELDKWASLWNWF-NH2 AC-YTSLIHSLIEESQ-X-QQE-XX-NEQELL-X-LDKWASLWNWF-NH2 AC-YTSLIHSLIEESQN-X-QEK-XX-EQELLE-X-DKWASLWNWF-NH2 AC-YTSLIHSLIEESQNQ-X-EKN-XX-QELLEL-X-KWASLWNWF-NH2 AC-YTSLIHSLIEESQNQQ-X-KNE-XX-ELLELD-X-WASLWNWF-NH2 AC-YTSLIHSLIEESQNQQE-X-NEQ-XX-LLELDK-X-ASLWNWF-NH2 AC-YTSLIHSLIEESQNQQEK-X-EQE-XX-LELDKW-X-SLWNWF-NH2 AC-YTSLIHSLIEESQNQQEKN-X-QEL-XX-ELDKWA-X-LWNWF-NH2 AC-YTSLIHSLIEESQNQQEKNE-X-ELL-XX-LDKWAS-X-WNWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQ-X-LLE-XX-DKWASL-X-NWF-NH2 Ac-YTS LIHSLIEESQNQQEKNEQE-X-LE L-XX-KWAS LW-X-W F- N H2 AC-YTSLIHSLIEESQNQQEKNEQEL-X-ELD-XX-WASLWN-X-F-NH2 AC-YTSLIHSLIEESQNQQEKNEQELL-X-LDK-XX-ASLWNW-X-NH2 AC-YT-X-LIHSLI-XX-ESQ-X-QQEKNEQELLELDKWASLWNWF-NH2 AC-YTS-X-IHSLIE-XX-SQN-X-QEKNEQELLELDKWASLWNWF-NH2 AC-YTSL-X-HSLIEE-XX-QNQ-X-EKNEQELLELDKWASLWNWF-NH2 AC-YTSLI-X-SLIEES-XX-NQQ-X-KNEQELLELDKWASLWNWF-NH2 AC-YTSLIH-X-LIEESQ-XX-QQE-X-NEQELLELDKWASLWNWF-NH2 AC-YTSLIHS-X-IEESQN-XX-QEK-X-EQELLELDKWASLWNWF-NH2 AC-YTSLIHSL-X-EESQNQ-XX-EKN-X-QELLELDKWASLWNWF-NH2 AC-YTSLIHSLI-X-ESQNQQ-XX-KNE-X-ELLELDKWASLWNWF-NH2 AC-YTSLIHSLIE-X-SQNQQE-XX-NEQ-X-LLELDKWASLWNWF-NH2 AC-YTSLIHSLIEE-X-QNQQEK-XX-EQE-X-LELDKWASLWNWF-NH2 AC-YTSLIHSLIEES-X-NQQEKN-XX-QEL-X-ELDKWASLWNWF-NH2 AC-YTSLIHSLIEESQ-X-QQEKNE-XX-ELL-X-LDKWASLWNWF-NH2 AC-YTSLIHSLIEESQN-X-QEKNEQ-XX-LLE-X-DKWASLWNWF-NH2 AC-YTSLIHSLIEESQNQ-X-EKNEQE-XX-LEL-X-KWASLWNWF-NH2 Ac-YTS LIHSLIEESQNQQ-X-KN EQ E L-XX- E L D-X-WAS LW N W F- N H 2 AC-YTSLIHSLIEESQNQQE-X-NEQELL-XX-LDK-X-ASLWNWF-NH2 AC-YTSLIHSLIEESQNQQEK-X-EQELLE-XX-DKW-X-SLWNWF-NH2 AC-YTSLIHSLIEESQNQQEKN-X-QELLEL-XX-KWA-X-LWNWF-NH2 AC-YTSLIHSLIEESQNQQEKNE-X-ELLELD-XX-WAS-X-WNWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQ-X-LLELDK-XX-ASL-X-NWF-NH2 AC-YTSLIHSLIEESQNQQEKNEQE-X-LELDKW-XX-SLW-X-WF-NH2
AC-YTSLIHSLIEESQNQQEKNEQEL-X-ELDKWA-XX-LWN-X-F-NH2
AC-YTSLIHSLIEESQNQQEKNEQELL-X-LDKWAS-XX-WNW-X-NH2
AC-YT-X-LIHSLI-XX-ESQNQQ-X-KNEQELLELDKWASLWNWF-NH2
AC-YTS-X-IHSLIE-XX-SQNQQE-X-NEQELLELDKWASLWNWF-NH2
AC-YTSL-X-HSLIEE-XX-QNQQEK-X-EQELLELDKWASLWNWF-NH2
AC-YTSLI-X-SLIEES-XX-NQQEKN-X-QELLELDKWASLWNWF-NH2
AC-YTSLIH-X-LIEESQ-XX-QQEKNE-X-ELLELDKWASLWNWF-NH2
AC-YTSLIHS-X-IEESQN-XX-QEKNEQ-X-LLELDKWASLWNWF-NH2
AC-YTSLIHSL-X-EESQNQ-XX-EKNEQE-X-LELDKWASLWNWF-NH2
AC-YTSLIHSLI-X-ESQNQQ-XX-KNEQEL-X-ELDKWASLWNWF-NH2
AC-YTSLIHSLIE-X-SQNQQE-XX-NEQELL-X-LDKWASLWNWF-NH2
AC-YTSLIHSLIEE-X-QNQQEK-XX-EQELLE-X-DKWASLWNWF-NH2
AC-YTSLIHSLIEES-X-NQQEKN-XX-QELLEL-X-KWASLWNWF-NH2
Ac-YTS LIHSLIEESQ-X-QQEKN E-XX- E L L E L D-X-WAS LW N W F-N H 2
AC-YTSLIHSLIEESQN-X-QEKNEQ-XX-LLELDK-X-ASLWNWF-NH2
AC-YTSLIHSLIEESQNQ-X-EKNEQE-XX-LELDKW-X-SLWNWF-NH2
AC-YTSLIHSLIEESQNQQ-X-KNEQEL-XX-ELDKWA-X-LWNWF-NH2
AC-YTSLIHSLIEESQNQQE-X-NEQELL-XX-LDKWAS-X-WNWF-NH2
AC-YTSLIHSLIEESQNQQEK-X-EQELLE-XX-DKWASL-X-NWF-NH2
AC-YTSLIHSLIEESQNQQEKN-X-QELLEL-XX-KWASLW-X-WF-NH2
AC-YTSLIHSLIEESQNQQEKNE-X-ELLELD-XX-WASLWN-X-F-NH2
AC-YTSLIHSLIEESQNQQEKNEQ-X-LLELDK-XX-ASLWNW-X-NH2
TABLE 2
Ac-NleTW-X-EWD-X-EIN-Z-YTS-Z-IHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EIN-Z-YTSLIH-Z-LIEESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINN-Z-TSL-Z-HSLIEESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINN-Z-TSLIHS-Z-IEESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNY-Z-SLI-Z-SLIEESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNY-Z-SLIHSL-Z-EESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYT-Z-LIH-Z-LIEESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYT-Z-LIHSLI-Z-ESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTS-Z-IHS-Z-IEESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTS-Z-IHSLIE-Z-SQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSL-Z-HSL-Z-EESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSL-Z-HSLIEE-Z-QNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSLI-Z-SLI-Z-ESQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSLI-Z-SLIEES-Z-NQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSLIH-Z-LIE-Z-SQNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSLIH-Z-LIEESQ-Z-QQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSLIHS-Z-IEE-Z-QNQQEKNEQELLE-NH2 Ac-NleTW-X-EWD-X-EINNYTSLIHS-Z-IEESQN-Z-QEKNEQELLE-NH2 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Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEE-Z-QNQQEK-Z-EQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEES-Z-NQQ-Z-KNEQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEES-Z-NQQEKN-Z-QELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQ-Z-QQE-Z-NEQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQ-Z-QQEKNE-Z-ELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQN-Z-QEK-Z-EQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQN-Z-QEKNEQ-Z-LLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQ-Z-EKN-Z-QELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQ-Z-EKNEQE-Z-LE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQQ-Z-KNE-Z-ELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQQ-Z-KNEQEL-Z-E-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQQE-Z-NEQ-Z-LLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQQE-Z-NEQELL-Z-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQQEK-Z-EQE-Z-LE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQQEKN-Z-QEL-Z-E-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-X-SLIEESQNQQEKNE-Z-ELL-Z-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHS-Z-IEE-Z-QNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHS-Z-IEESQN-Z-QEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSL-Z-EES-Z-NQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSL-Z-EESQNQ-Z-EKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLI-Z-ESQ-Z-QQEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLI-Z-ESQNQQ-Z-KNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIE-Z-SQN-Z-QEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIE-Z-SQNQQE-Z-NEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEE-Z-QNQ-Z-EKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEE-Z-QNQQEK-Z-EQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEES-Z-NQQ-Z-KNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEES-Z-NQQEKN-Z-QELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQ-Z-QQE-Z-NEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQ-Z-QQEKNE-Z-ELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQN-Z-QEK-Z-EQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQN-Z-QEKNEQ-Z-LLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQ-Z-EKN-Z-QELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQ-Z-EKNEQE-Z-LE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQQ-Z-KNE-Z-ELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQQ-Z-KNEQEL-Z-E-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQQE-Z-NEQ-Z-LLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQQE-Z-NEQELL-Z-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQQEK-Z-EQE-Z-LE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQQEKN-Z-QEL-Z-E-NH2 Ac-NleTWNIeEWDREIN-X-YTS-X-IHSLIEESQNQQEKNE-Z-ELL-Z-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIE-Z-SQN-Z-QEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIE-Z-SQNQQE-Z-NEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEE-Z-QNQ-Z-EKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEE-Z-QNQQEK-Z-EQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEES-Z-NQQ-Z-KNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEES-Z-NQQEKN-Z-QELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQ-Z-QQE-Z-NEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQ-Z-QQEKNE-Z-ELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQN-Z-QEK-Z-EQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQN-Z-QEKNEQ-Z-LLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQ-Z-EKN-Z-QELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQ-Z-EKNEQE-Z-LE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQQ-Z-KNE-Z-ELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQQ-Z-KNEQEL-Z-E-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQQE-Z-NEQ-Z-LLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQQE-Z-NEQELL-Z-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQQEK-Z-EQE-Z-LE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQQEKN-Z-QEL-Z-E-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-X-LIEESQNQQEKNE-Z-ELL-Z-NH2 Ac-NleTW-X-EWD-XX-EIN-X-YTSLIHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIe-X-WDR-XX-INN-X-TSLIHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeE-X-DRE-XX-NNY-X-SLIHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEW-X-REI-XX-NYT-X-LIHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWD-X-EIN-XX-YTS-X-IHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDR-X-INN-XX-TSL-X-HSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDRE-X-NNY-XX-SLI-X-SLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYT-XX-LIH-X-LIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-XX-IHS-X-IEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINN-X-TSL-XX-HSL-X-EESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNY-X-SLI-XX-SLI-X-ESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYT-X-LIH-XX-LIE-X-SQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTS-X-IHS-XX-IEE-X-QNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSL-X-HSL-XX-EES-X-NQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLI-X-SLI-XX-ESQ-X-QQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIH-X-LIE-XX-SQN-X-QEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHS-X-IEE-XX-QNQ-X-EKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSL-X-EES-XX-NQQ-X-KNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLI-X-ESQ-XX-QQE-X-NEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIE-X-SQN-XX-QEK-X-EQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEE-X-QNQ-XX-EKN-X-QELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEES-X-NQQ-XX-KNE-X-ELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEESQ-X-QQE-XX-NEQ-X-LLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEESQN-X-QEK-XX-EQE-X-LE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEESQNQ-X-EKN-XX-QEL-X-E-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEESQNQQ-X-KNE-XX-ELL-X-NH2 Ac-NleTW-X-EWD-XX-EINNYT-X-LIHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIe-X-WDR-XX-INNYTS-X-IHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeE-X-DRE-XX-NNYTSL-X-HSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEW-X-REI-XX-NYTSLI-X-SLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWD-X-EIN-XX-YTSLIH-X-LIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDR-X-INN-XX-TSLIHS-X-IEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDRE-X-NNY-XX-SLIHSL-X-EESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYT-XX-LIHSLI-X-ESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTS-XX-IHSLIE-X-SQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINN-X-TSL-XX-HSLIEE-X-QNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNY-X-SLI-XX-SLIEES-X-NQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYT-X-LIH-XX-LIEESQ-X-QQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTS-X-IHS-XX-IEESQN-X-QEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSL-X-HSL-XX-EESQNQ-X-EKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLI-X-SLI-XX-ESQNQQ-X-KNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIH-X-LIE-XX-SQNQQE-X-NEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHS-X-IEE-XX-QNQQEK-X-EQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSL-X-EES-XX-NQQEKN-X-QELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLI-X-ESQ-XX-QQEKNE-X-ELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIE-X-SQN-XX-QEKNEQ-X-LLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEE-X-QNQ-XX-EKNEQE-X-LE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEES-X-NQQ-XX-KNEQEL-X-E-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEESQ-X-QQE-XX-NEQELL-X-NH2 Ac-NleTW-X-EWDREI-XX-NYT-X-LIHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIe-X-WDREIN-XX-YTS-X-IHSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeE-X-DREINN-XX-TSL-X-HSLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEW-X-REINNY-XX-SLI-X-SLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWD-X-EINNYT-XX-LIH-X-LIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDR-X-INNYTS-XX-IHS-X-IEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDRE-X-NNYTSL-XX-HSL-X-EESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-XX-SLI-X-ESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-XX-LIE-X-SQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINN-X-TSLIHS-XX-IEE-X-QNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNY-X-SLIHSL-XX-EES-X-NQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYT-X-LIHSLI-XX-ESQ-X-QQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTS-X-IHSLIE-XX-SQN-X-QEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSL-X-HSLIEE-XX-QNQ-X-EKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLI-X-SLIEES-XX-NQQ-X-KNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIH-X-LIEESQ-XX-QQE-X-NEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHS-X-IEESQN-XX-QEK-X-EQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSL-X-EESQNQ-XX-EKN-X-QELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLI-X-ESQNQQ-XX-KNE-X-ELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIE-X-SQNQQE-XX-NEQ-X-LLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEE-X-QNQQEK-XX-EQE-X-LE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEES-X-NQQEKN-XX-QEL-X-E-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIEESQ-X-QQEKNE-XX-ELL-X-NH2 Ac-NleTW-X-EWDREI-XX-NYTSLI-X-SLIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIe-X-WDREIN-XX-YTSLIH-X-LIEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeE-X-DREINN-XX-TSLIHS-X-IEESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEW-X-REINNY-XX-SLIHSL-X-EESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWD-X-EINNYT-XX-LIHSLI-X-ESQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDR-X-INNYTS-XX-IHSLIE-X-SQNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDRE-X-NNYTSL-XX-HSLIEE-X-QNQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREI-X-NYTSLI-XX-SLIEES-X-NQQEKNEQELLE-NH2 Ac-NleTWNIeEWDREIN-X-YTSLIH-XX-LIEESQ-X-QQEKNEQELLE-NH2 Ac-NleTWNIeEWDREINN-X-TSLIHS-XX-IEESQN-X-QEKNEQELLE-NH2 Ac-NleTWNIeEWDREINNY-X-SLIHSL-XX-EESQNQ-X-EKNEQELLE-NH2 Ac-NleTWNIeEWDREINNYT-X-LIHSLI-XX-ESQNQQ-X-KNEQELLE-NH2 Ac-NleTWNIeEWDREINNYTS-X-IHSLIE-XX-SQNQQE-X-NEQELLE-NH2 Ac-NleTWNIeEWDREINNYTSL-X-HSLIEE-XX-QNQQEK-X-EQELLE-NH2 Ac-NleTWNIeEWDREINNYTSLI-X-SLIEES-XX-NQQEKN-X-QELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIH-X-LIEESQ-XX-QQEKNE-X-ELLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHS-X-IEESQN-XX-QEKNEQ-X-LLE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSL-X-EESQNQ-XX-EKNEQE-X-LE-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLI-X-ESQNQQ-XX-KNEQEL-X-E-NH2 Ac-NleTWNIeEWDREINNYTSLIHSLIE-X-SQNQQE-XX-NEQELL-X-NH2
Peptidomimetic Macrocycles of the Invention
Peptidomimetic Macrocycles I
1. Provided herein is a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1, 2 and 3, wherein the peptidomimetic macrocycle has an IC50 of about 10000 nM or less against a target and in cells selected from the group consisting of: target: HIV-1 IIIB, cells: CEM-SS; target: HIV-1NL4-3, cells: MT-4; and target: HIV-1MDR769, cells: hPBMC, wherein the peptidomimetic macrocycle is not a compound having the sequence:
(SP-13)YT$LI H$LIEESQNQQEKNEQELLEL$KW A $ L
W NWF,
(SP-33) MeTWSEWD $EINNYTSLIHSL IEESQN$QEK$EQE L L E, and
SP-41)NleTW$EWD$EINNYT$LIH$LI EESQNQ$E KN$ Q ELLE.
[0065] HIV gp41 is discussed, for example, in International Application Publication No. WO 2009/018261. [0066] The sequence of the peptidomimetic macrocycle can be, for example, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1 , 2 and 3. The sequence of the peptidomimetic macrocycle can be selected from the group consisting of the amino acid sequences in Tables 1 , 2, and 3.
[0067] The target can be HIV-1 IIIB and cells can be CEM-SS.
[0068] The target can be HIV-1NL4-3 and cells can be MT-4.
[0069] The target can be HIV- 1 MDR769 and the cells can be hPBMC .
[0070] The peptidomimetic macrocycle can have an IC50, for example, of about 10000 nM or less, about 6000 nM or less, about 3000 nM or less, about 1000 nM or less, about 500 nM or less, about 100 nM or less, about 10 nM or less, about 5 nM or less, about 4 nM or less, about 3nM or less, about 2 nM or less, about 1 nM or less, or about 0.5 nM or less, against a target and in cells selected from the group consisting of target: HIV-1 IIIB, cells: CEM-SS; target: HIV-1NL4-3, cells: MT-4; and target: HIV-1MDR769, cells: hPBMC.
[0071] The peptidomimetic macrocycle can have an IC50 of, for example, about 10 nM or less, about 5 nM or less, about 4 nM or less, about 3nM or less, about 2 nM or less, about 1 nM or less, or about 0.5 nM or less, when the target is HIV-1MDR769 and the cells are hPBMC.
[0072] The peptidomimetic macrocycle can have a positive number of amino acids, for example, of 60 or less, or 50 or less, of 40 or less, of 30 or less, or of 20 of less.
[0073] The peptidomimetic macrocycle can comprise one or more independent crosslinkers, each crosslinker independently linking the a-positions of two amino acids of the peptidomimetic macrocycle.
[0074] In the peptidomimetic macrocycle, at least one of the two amino acids can be an α,α- disubstituted amino acid.
[0075] The peptidomimetic macrocycle can comprise one crosslinker linking the α-positions of two amino acids of the peptidomimetic macrocycle.
[0076] The peptidomimetic macrocycle can comprise two independent crosslinkers, each
crosslinker independently linking the α-positions of two amino acids of the
peptidomimetic macrocycle.
Peptidomimetic Macrocycles II [0077] Provided herein is a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to HIV gp41626-663, further comprising at least two independent crosslinkers, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid, wherein the first amino acid and the second amino acid are selected from the group consisting of amino acids: 629 and 633; 630 and 634; 632 and 636; and 633 and 637; wherein the third amino acid and the fourth amino acid are selected from the group consisting of amino acids: 650 and 654; 651 and 655; 652 and 656; 653 and 657; 654 and 658; 655 and 659; 656 and 660; and 657 and 771; with the proviso that when the first crosslinker connects amino acids pairs 629 and 633 and the second crosslinker connects amino acid pair 653 and 657, there is an additional crosslinker that connects amino acids pairs 641 and 645 or 642 and 646.
[0078] The peptidomimetic macrocycle can be at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to HIV gp41626-663.
[0079] The peptidomimetic macrocycle can comprise an additional cross linker, wherein the additional crosslinker connects an amino acid pair selected from the group consisting of: 640 and 644; 641 and 645; and 642 and 646.
[0080] In the peptidomimetic macrocycle, the first crosslinker can connect amino acid pair 629 and 633, and the second crosslinker can connect amino acid pair 654 and 658. When this happens, the peptidomimetic macrocycle can have a sequence that is at least about 60% identical to the sequence:
Nle TW$EWD$EINNYTSLIHSLIEESQNQQ$KNE$ELLE
(SP-36).
The sequence can be at least about 60%>, at least about 70%>, at least about 80%>, at least about 85%o, at least about 90%>, or at least about 95% identical to SP-36.
[0081] In the peptidomimetic macrocycle, the first crosslinker connect amino acid pair 629 and
633, and the second crosslinker can connect amino acid pair 655 and 659. When this happens, the peptidomimetic macrocycle can have a sequence that is at least about 60% identical to the sequence:
Nle TW$EWD$EINNYTSLIHSLIEESQNQQE$NEQ$LLE
(SP-37).
The sequence can be at least about 60%>, at least about 70%>, at least about 80%>, at least about 85%o, at least about 90%>, or at least about 95% identical to SP-37. [0082] In the peptidomimetic macrocycle, the first crosslinker can connect amino acid pair 633 and 637, and the second crosslinker can connect amino acid pair 655 and 659. When this happens, the peptidomimetic macrocycle can have a sequence that is at least about 60% identical to the sequence:
Nle T W Nle E W D $ E I N $ Y T S L I H S L I E E S Q N Q Q E $ N E Q $ L L
E (SP-48).
The sequence can be at least about 60%>, at least about 70%>, at least about 80%>, at least about 85%o, at least about 90%>, or at least about 95% identical to SP-34.
[0083] The peptidomimetic macrocycle can comprise a helix.
[0084] The peptidomimetic macrocycle can comprise an alpha-helix.
[0085] The peptidomimetic macrocycle can comprise an α,α-disubstituted amino acid.
[0086] The peptidomimetic macrocycle can be such that at least one of the two amino acids in one or more amino acid pairs is an α,α-disubstituted amino acid.
Peptidomimetic Macrocycles III
[0087] Also provided herein is a peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60%> identical to HIV gp41 638-673, further having one crosslinker, wherein the crosslinker connects a first amino acid to a second amino acid, wherein the first amino acid and the second amino acid are 652 and 659. The
peptidomimetic can be, for example, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% 60% identical to HIV gp41 638-673.
[0088] The peptidomimetic macrocycle can comprise a helix.
[0089] The peptidomimetic macrocycle can comprise an alpha-helix.
[0090] The peptidomimetic macrocycle can comprise an α,α-disubstituted amino acid.
[0091] The peptidomimetic macrocycle can be such that at least one of the two amino acids in one or more amino acid pairs is an α,α-disubstituted amino acid.
Peptidomimetic Macrocycles IV
[0092] Further provided herein is a peptidomimetic macrocycle comprising an amino acid
sequence which is at least about 60%> identical to HIV gp41 638-673, further comprising at least two independent crosslinkers connecting at least two pairs of amino acids, wherein the first crosslmker connects a first amino acid to a second amino acid, and the second crosslmker connects a third amino acid to a fourth amino acid, wherein each pair of first and second amino acids are selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671;wherein when the first crosslmker connects amino acid pair 640 and 644, the second crosslmker does not connect the amino acid pair 664 and 668 or 666 and 670; and wherein when the first crosslmker connects amino acid pair 653 and 657, the second crosslmker does not connect amino acid pair 666 and 670.
The peptidomimetic macrocycle can be at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to HIV gp41 638-673.
[0093] In the peptidomimetic macrocycle of claim 21, the first crosslmker can connect an amino acid pair selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; and 653 and 657.
[0094] In the peptidomimetic macrocycle, the second crosslmker can connecxt an amino acid pair selected from the group consisting of: 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671.
[0095] The peptidomimetic macrocycle can comprise a helix.
[0096] The peptidomimetic macrocycle can comprise an alpha-helix.
[0097] The peptidomimetic macrocycle can comprise an α,α-disubstituted amino acid.
[0098] The peptidomimetic macrocycle can be such that at least one of the two amino acids in one or more amino acid pairs is an α,α-disubstituted amino acid.
Formulae [0099] In any emboidment herein, unless otherwise indicated, the peptidomimetic macrocycle can be of Formula (I):
Figure imgf000041_0001
Formula I
wherein:
each A, C, D, and E is independently a natural or non-natural amino acid;
B is a natural or non-natural amino acid, amino acid analog,
Figure imgf000041_0002
, [-NH-L3-
CO-], [-NH-L3-S02-], or [- H-L3-];
Ri and R2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is unsubstituted or substituted with halo-;
R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which except hydrogen is optionally substituted with R5;
L is a macrocycle-forming linker of the formula -Li-L2-;
Li and L2 and L3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R4-]n, each being optionally substituted with R5;
each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene;
each K is O, S, SO, S02, CO, C02, or CONR3;
each R5 is independently halogen, alkyl, -OR6, -N(R6)2, -SR6, -SOR5, -S02R6, -C02R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
each R6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; R7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R5, or part of a cyclic structure with a D residue;
R8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R5, or part of a cyclic structure with an E residue;
v and w are independently integers from 1-1000, for example: 1-500, 1-300, 1-200, 1- 100, 1-50, 1-40, 1-30, 1-20, 1- 10;
u is an integer from 1-10, for example 1, 2, or 3;
x, y and z are independently integers from 0-10; for example, the sum of x+y+z can equal 2, 3 or 6, for example, 3 or 6; and
n is an integer from 1-5.
[00100] In one example, at least one of Ri and R2 is alkyl, unsubstituted or substituted with halo-.
In another example, both Ri and R2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of Ri and R2 is methyl. In other embodiments, Ri and R2 are methyl.
[00101] In some embodiments of the invention, x+y+z is at least 3. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected. For example, a sequence represented by the formula [A]x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gin-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges. Similarly, when u is greater than 1, each compound of the invention may encompass peptidomimetic macrocycles which are the same or different. For example, a compound of the invention may comprise
peptidomimetic macrocycles comprising different linker lengths or chemical
compositions.
[00102] In some embodiments, the peptidomimetic macrocycle of the invention comprises a
secondary structure which is an a-helix and Rs is -H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α-disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments, at least one of
Figure imgf000043_0001
[00103] In other embodiments, the length of the macrocycle-forming linker L as measured from a first Ca to a second Ca is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Ca to a second Ca.
[00104] In one embodiment, the peptidomimetic macrocycle of Formula (I) is:
Figure imgf000043_0002
[00105] wherein each Ri and R2 is independently independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-.
[00106] In related embodiments, the peptidomimetic macrocycle of Formula (I) is:
Figure imgf000043_0003
or
Figure imgf000043_0004
[00107] In other embodiments, the peptidomimetic macrocycle of Formula (I) is a compound of any of the formulas shown below:
Figure imgf000044_0001
-43-
Figure imgf000045_0001
Figure imgf000045_0002
Figure imgf000045_0003
Figure imgf000045_0004
Figure imgf000045_0005
Figure imgf000045_0006
Figure imgf000046_0001
wherein "AA" represents any natural or non-natural amino acid side chain and " is [D]v, [E]v as defined above, and n is an integer between 0 and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. In other embodiments, n is less than 50.
[00108] Exemplary embodiments of the macrocycle-forming linker L are shown below.
where X, Y = -CH2 where X, Y = -CH2
m, n, o, p = 0-10 m, n, o, p = 0-10
Figure imgf000046_0003
where X, Y = -CH2-, O, S, or NH where X, Y = -CH2-, O, S, or NH m, n, o, p = 0-10 m, n, o = 0-10
R = H, alkyl, other substituent
[00109] In some embodiments, L is a macrocycle-forming linker of the formula
Figure imgf000046_0004
[00110] Exemplary embodiments of such macrocycle-forming linkers L are shown below.
Figure imgf000047_0001
Figure imgf000048_0001
-47- [00111] In other embodiments, D and/or E in the compound of Formula I are further modified in order to facilitate cellular uptake. In some embodiments, lipidating or PEGylating a peptidomimetic macrocycle facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.
[00112] In other embodiments, at least one of [D] and [E] in the compound of Formula I
represents a moiety comprising an additional macrocycle-forming linker such that the peptidomimetic macrocycle comprises at least two macrocycle-forming linkers. In a specific embodiment, a peptidomimetic macrocycle comprises two macrocycle-forming linkers.
[00113] In the peptidomimetic macrocycles of the invention, any of the macrocycle-forming
linkers described herein may be used in any combination with any of the sequences shown in Tables 1-6 and also with any of the R- substituents indicated herein.
[00114] In some embodiments, the peptidomimetic macrocycle comprises at least one a-helix motif. For example, A, B and/or C in the compound of Formula I include one or more a- helices. As a general matter, a-helices include between 3 and 4 amino acid residues per turn. In some embodiments, the a-helix of the peptidomimetic macrocycle includes 1 to 5 turns and, therefore, 3 to 20 amino acid residues. In specific embodiments, the a-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In some embodiments, the
macrocycle-forming linker stabilizes an a-helix motif included within the peptidomimetic macrocycle. Thus, in some embodiments, the length of the macrocycle-forming linker L from a first Ca to a second Ca is selected to increase the stability of an a-helix. In some embodiments, the macrocycle-forming linker spans from 1 turn to 5 turns of the a-helix. In some embodiments, the macrocycle-forming linker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns of the a-helix. In some embodiments, the length of the macrocycle-forming linker is approximately 5 A to 9 A per turn of the a-helix, or approximately 6 A to 8 A per turn of the a-helix. Where the macrocycle-forming linker spans approximately 1 turn of an a-helix, the length is equal to approximately 5 carbon- carbon bonds to 13 carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11 carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where the macrocycle- forming linker spans approximately 2 turns of an a-helix, the length is equal to approximately 8 carbon-carbon bonds to 16 carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14 carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 3 turns of an a-helix, the length is equal to approximately 14 carbon-carbon bonds to 22 carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20 carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 4 turns of an a-helix, the length is equal to approximately 20 carbon-carbon bonds to 28 carbon- carbon bonds, approximately 22 carbon-carbon bonds to 26 carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where the macrocycle-forming linker spans approximately 5 turns of an a-helix, the length is equal to approximately 26 carbon- carbon bonds to 34 carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32 carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where the macrocycle- forming linker spans approximately 1 turn of an a-helix, the linkage contains
approximately 4 atoms to 12 atoms, approximately 6 atoms to 10 atoms, or
approximately 8 atoms. Where the macrocycle-forming linker spans approximately 2 turns of the a-helix, the linkage contains approximately 7 atoms to 15 atoms,
approximately 9 atoms to 13 atoms, or approximately 11 atoms. Where the macrocycle- forming linker spans approximately 3 turns of the a-helix, the linkage contains approximately 13 atoms to 21 atoms, approximately 15 atoms to 19 atoms, or approximately 17 atoms. Where the macrocycle-forming linker spans approximately 4 turns of the a-helix, the linkage contains approximately 19 atoms to 27 atoms, approximately 21 atoms to 25 atoms, or approximately 23 atoms. Where the macrocycle- forming linker spans approximately 5 turns of the a-helix, the linkage contains approximately 25 atoms to 33 atoms, approximately 27 atoms to 31 atoms, or approximately 29 atoms. Where the macrocycle-forming linker spans approximately 1 turn of the a-helix, the resulting macrocycle forms a ring containing approximately 17 members to 25 members, approximately 19 members to 23 members, or approximately 21 members. Where the macrocycle-forming linker spans approximately 2 turns of the a- helix, the resulting macrocycle forms a ring containing approximately 29 members to 37 members, approximately 31 members to 35 members, or approximately 33 members. Where the macrocycle-forming linker spans approximately 3 turns of the a-helix, the resulting macrocycle forms a ring containing approximately 44 members to 52 members, approximately 46 members to 50 members, or approximately 48 members. Where the macrocycle-forming linker spans approximately 4 turns of the a-helix, the resulting macrocycle forms a ring containing approximately 59 members to 67 members, approximately 61 members to 65 members, or approximately 63 members. Where the macrocycle-forming linker spans approximately 5 turns of the a-helix, the resulting macrocycle forms a ring containing approximately 74 members to 82 members, approximately 76 members to 80 members, or approximately 78 members.
[00115] In other embodiments, the peptidomimetic macrocycle is of Formula (II) or (Ha):
Figure imgf000051_0001
11 Formula (Ila), wherein:
each A, C, D, and E is independently a natural or non-natural amino acid;
Figure imgf000051_0002
B is a natural or non-natural amino acid, amino acid analog, O , [-NH-L3-
CO-], [-NH-L3-SO2-], or [-NH-L3-];
Ri and R2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;
R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which except hydrogen is optionally substituted with R5;
Li and L2 and L3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R4-]n, each being optionally substituted with R5;
each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene;
each K is O, S, SO, S02, CO, C02, or CONR3; each R5 is independently halogen, alkyl, -OR6, -N(R6)2, -SR6, -SOR5, -S02R6, -C02R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
each R6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
R7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R5;
v and w are independently integers from 1-1000;
u is an integer from 1-10, for example 1, 2, or 3;
x, y and z are independently integers from 0-10; for example, the sum of x+y+z can equal
2, 3 or 6, for example, 3 or 6; and
n is an integer from 1-5.
[00116] In one example, at least one of Ri and R2 is alkyl, unsubstituted or substituted with halo-.
In another example, both Ri and R2 are independently alkyl, unsubstituted or substituted with halo-. In some embodiments, at least one of Ri and R2 is methyl. In other embodiments, Ri and R2 are methyl.
[00117] In some embodiments of the invention, x+y+z is at least 1. In other embodiments of the invention, x+y+z is at least 2. In other embodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each occurrence of A, B, C, D or E in a macrocycle or macrocycle precursor of the invention is independently selected. For example, a sequence represented by the formula [A]x, when x is 3, encompasses embodiments where the amino acids are not identical, e.g. Gin-Asp-Ala as well as embodiments where the amino acids are identical, e.g. Gln-Gln-Gln. This applies for any value of x, y, or z in the indicated ranges.
[00118] In some embodiments, the peptidomimetic macrocycle of the invention comprises a
secondary structure which is an a-helix and Rs in Formula (I) is -H, allowing intrahelical hydrogen bonding. In some embodiments, at least one of A, B, C, D or E is an α,α- disubstituted amino acid. In one example, B is an α,α-disubstituted amino acid. For instance, at least one of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,
at least one of A, B, C, D or E is
Figure imgf000052_0001
.
[00119] In other embodiments, the length of the macrocycle-forming linker L as measured from a first Ca to a second Ca is selected to stabilize a desired secondary peptide structure, such as an a-helix formed by residues of the peptidomimetic macrocycle including, but not necessarily limited to, those between the first Ca to a second Ca.
[00120] Exemplary embodiments of the macrocycle-forming linker -Li-L2- are shown below.
Figure imgf000053_0001
where X, Y = -CH2 where X, Y = -CH2
m, n, o, p = 0-10 m, n, o, p = 0-10
Figure imgf000053_0002
where X, Y = -CH2- O, S, or NH where X, Y = -CH2- O, S, or NH m, n, o, p = 0-10 m, n, o = 0-10
R = H, alkyl, other substituent
[00121] In some embodiments, L is a macrocycle-forming linker of the formula
Figure imgf000053_0003
[00122] Exemplary embodiments of such macrocycle-forming linkers L are shown below.
Figure imgf000054_0001
-53-
Figure imgf000055_0001
-54- [00123] In some embodiments, unless otherwise indicated, the peptidomimetic macrocycle can have the Formula III):
Figure imgf000056_0001
wherein each A, C, D, and E is independently an amino acid;
B is an amino acid,
Figure imgf000056_0002
, [-NH-L3-CO-], [-NH-L3-SO2-], or [-NH-L3-];
L' is a macrocycle-forming linker of the formula -Li'-L2'-;
and wherein A, B, C, D, and E, taken together with the crosslinked amino acids connected by the macrocycle-forming linkers L and L', form the amino acid sequence of the peptidomimetic macrocycle;
Ri' and R2' are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
Li ' and L2' are independently alkylene, alkenylene, alkynyl ene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R4-]n, each being optionally substituted with R5;
each K is independently O, S, SO, S02, CO, C02, or CONR3;
R7' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with a D residue;
Rs' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with an E residue;
v' and w' are independently integers from 1-100;
x', y' and z' are independently integers from 0-10; and
n is an integer from 1-5.
Methods of Use
[00124] In one aspect, the present invention provides novel peptidomimetic macrocycles that are useful in competitive binding assays to identify agents which bind to the natural ligand(s) of the proteins or peptides upon which the peptidomimetic macrocycles are modeled. For example, in the HIV gp41 system, labeled peptidomimetic macrocycles based on HIV gp41 can be used in a binding assay along with small molecules that competitively bind to the HIV gp41 six helix bundle assembly or sub-complexes thereof. Competitive binding studies allow for rapid in vitro evaluation and determination of drug candidates specific for the HIV gp41 system. Such binding studies may be performed with any of the peptidomimetic macrocycles disclosed herein and their binding partners.
[00125] The invention further provides for the generation of antibodies against the
peptidomimetic macrocycles herein. In some embodiments, these antibodies specifically bind both the peptidomimetic macrocycle and the precursor peptides, such as HIV gp41, to which the peptidomimetic macrocycles are related. Such antibodies, for example, disrupt the native protein-protein interactions, for example, in the HIV gp41 fusion complex.
[00126] Also provided is a method of preventing HIV infection in a subject comprising
administering to the subject a peptidomimetic macrocycle herein.
[00127] Additionally provided is a method of inhibiting HIV viral entry in a subject comprising administering to the subject a peptidomimetic macrocycle herein.
[00128] Further provided is a method of treating HIV infection and associated
immunodeficiencies in a subject comprising administering to the subject a
peptidomimetic macrocycle herein.
[00129] Also provided is a method of inhibiting HIV viral entry in a subject comprising
administering to the subject a peptidomimetic macrocycle herein.
[00130] Additionally provided is a method of antagonizing the formation of the six-helix bundle
HIV HIV gp41 fusion complex in a subject comprising administering to the subject a peptidomimetic macrocycle herein.
Further provided is a method of preventing or treating HIV infection and associated immunodeficiencies (i.e., AIDS) in a subject comprising administering an antagonist of the six -helix bundle HIV HIV gp41 fusion complex, wherein the antagonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week, wherein the antagonist can be a peptidomimetic macrocycle herein.
[00131] Also provided is a method of preventing or treating HIV infection and associated
immunodeficiencies (i.e., AIDS) in a subject comprising administering an antagonist of the six -helix bundle HIV HIV gp41 fusion complex, wherein the frequency or intensity of the site of injection reaction is reduced relative to that of enfuvirtide. The antagonist can be any peptidomimetic macrocycle herein. In any method therein, the subject can be a subject in need thereof (e.g., in need of treatment or prevention or antagonizing). Any method herein can comprise administering an effective amount of a peptidomimetic macrocyle herein or compound of the invention herein to a warm blooded animal, including a human.
[00132] In other aspects, the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with HIV infection.
[00133] In another aspect, the present invention provides methods for treating or preventing a disease including HIV infection and associated immunodeficiencies (i.e., AIDS) by interfering with the assembly of the six helix bundle HIV gp41 fusion complex.
[00134] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.
Preparation of Peptidomimetic Macrocycles
[00135] Peptidomimetic macrocycles of the invention may be prepared by any of a variety of methods known in the art. For example, any of the residues indicated by "X" in Tables 1, 2 or 3 may be substituted with a residue capable of forming a crosslinker with a second residue in the same molecule or a precursor of such a residue.
[00136] Various methods to effect formation of peptidomimetic macrocycles are known in the art.
For example, the preparation of peptidomimetic macrocycles of Formula I is described in Schafmeister et al, J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al, Science 305: 1466-1470 (2004); US Patent No. 7,192,713 and PCT application WO 2008/121767. The α,α-disubstituted amino acids and amino acid precursors disclosed in the cited references may be employed in synthesis of the peptidomimetic macrocycle precursor polypeptides. For example, the "S5-olefm amino acid" is (S)-a-(2'-pentenyl) alanine and the "R8 olefin amino acid" is (R)-a-(2'-octenyl) alanine. Following incorporation of such amino acids into precursor polypeptides, the terminal olefins are reacted with a metathesis catalyst, leading to the formation of the peptidomimetic macrocycle.
[00137] In other embodiments, the peptidomimetic macrocyles of the invention are of Formula II or Ila. Methods for the preparation of such macrocycles are described, for example, in US Patent No. 7,202,332.
[00138] Additional methods of forming peptidomimetic macrocycles which are envisioned as suitable to perform the present invention include those disclosed by Mustapa, M. Firouz Mohd et al, J. Org. Chem (2003), 68, pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp. 1403-1406; U.S. Patent No. 5,364,851 ; U.S. Patent No. 5,446, 128; U.S. Patent No. 5,824,483; U.S. Patent No. 6,713,280; and U.S. Patent No. 7,202,332. In such embodiments, aminoacid precursors are used containing an additional substituent R- at the alpha position. Such aminoacids are incorporated into the macrocycle precursor at the desired positions, which may be at the positions where the crosslinker is substituted or, alternatively, elsewhere in the sequence of the macrocycle precursor. Cyclization of the precursor is then performed according to the indicated method.
Assays
[00139] The properties of the peptidomimetic macrocycles of the invention are assayed, for
example, by using the methods described below. In some embodiments, a
peptidomimetic macrocycle of the invention has improved biological properties relative to a corresponding polypeptide lacking the substituents described herein.
Assay to Determine a-helicity.
[00140] In solution, the secondary structure of polypeptides with a-helical domains will reach a dynamic equilibrium between random coil structures and α-helical structures, often expressed as a "percent helicity". Thus, for example, alpha-helical domains are predominantly random coils in solution, with α-helical content usually under 25%.
Peptidomimetic macrocycles with optimized linkers, on the other hand, possess, for example, an alpha-helicity that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide. In some embodiments, macrocycles of the invention will possess an alpha-helicity of greater than 50%. To assay the helicity of peptidomimetic macrocyles of the invention, the compounds are dissolved in an aqueous solution (e.g. 50 mM potassium phosphate solution at pH 7, or distilled H20, to concentrations of 25-50 μΜ). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J- 710) using standard measurement parameters (e.g., temperature, 20°C; wavelength, 190- 260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The a-helical content of each peptide is calculated by dividing the mean residue ellipticity (e.g., [< ]222obs) by the reported value for a model helical decapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).
Assay to Determine Melting Temperature (Tm .
[00141] A peptidomimetic macrocycle of the invention comprising a secondary structure such as an a-helix exhibits, for example, a higher melting temperature than a corresponding uncrosslinked polypeptide. Typically peptidomimetic macrocycles of the invention exhibit Tm of > 60°C representing a highly stable structure in aqueous solutions. To assay the effect of macrocycle formation on meltine temperature, peptidomimetic macrocycles or unmodified peptides are dissolved in distilled H20 (e.g. at a final concentration of 50 μΜ) and the Tm is determined by measuring the change in ellipticity over a temperature range (e.g,. 4 to 95 °C) on a spectropolarimeter (e.g., Jasco J-710) using standard parameters (e.g. wavelength 222nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: l°C/min; path length, 0.1 cm).
Protease Resistance Assay.
[00142] The amide bond of the peptide backbone is susceptible to hydrolysis by proteases,
thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries the amide backbone and therefore may shield it from proteolytic cleavage. The peptidomimetic macrocycles of the present invention may be subjected to in vitro trypsin proteolysis to assess for any change in degradation rate compared to a corresponding uncrosslinked polypeptide. For example, the peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycle and peptidomimetic precursor (5 meg) are incubated with trypsin agarose (Pierce) (S/E -125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC -based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of ln[S] versus time (k=-lXslope). Ex Vivo Stability Assay.
[00143] Peptidomimetic macrocycles with optimized linkers possess, for example, an ex vivo half-life that is at least two-fold greater than that of a corresponding uncrosslinked polypeptide, and possess an ex vivo half-life of 12 hours or more. For ex vivo serum stability studies, a variety of assays may be used. For example, a peptidomimetic macrocycle and a corresponding uncrosslinked polypeptide (2 meg) are incubated with fresh mouse, rat and/or human serum (2 mL) at 37°C for 0, 1, 2, 4, 8, and 24 hours. To determine the level of intact compound, the following procedure may be used: The samples are extracted by transferring 100 μΐ of sera to 2 ml centrifuge tubes followed by the addition of 10 of 50 % formic acid and 500μί acetonitrile and centrifugation at 14,000 RPM for 10 min at 4 ± 2°C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N2 < 10 psi, 37°C. The samples are
reconstituted in ΙΟΟμί of 50:50 acetonitrile: water and submitted to LC-MS/MS analysis.
In vitro Binding Assays.
[00144] To assess the binding and affinity of peptidomimetic macrocycles and peptidomimetic precursors to acceptor proteins, a fluorescence polarization assay (FPA) isused, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers {e.g., FITC) attached to molecules with high apparent molecular weights {e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules {e.g. FITC- labeled peptides that are free in solution).
[00145] For example, fluoresceinated peptidomimetic macrocycles (25 nM) are incubated with the acceptor protein (25- lOOOnM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Binding activity ismeasured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA). A peptidomimetic macrocycle of the invention shows, in some instances, similar or lower Kd than a corresponding uncrosslinked polypeptide. In vitro Displacement Assays To Characterize Antagonists of Peptide-Protein
Interactions.
[00146] To assess the binding and affinity of compounds that antagonize the interaction between a peptide and an acceptor protein, a fluorescence polarization assay (FPA) utilizing a fluoresceinated peptidomimetic macrocycle derived from a peptidomimetic precursor sequence is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers {e.g., FITC) attached to molecules with high apparent molecular weights {e.g. FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules {e.g. FITC-labeled peptides that are free in solution). A compound that antagonizes the interaction between the fluoresceinated peptidomimetic macrocycle and an acceptor protein will be detected in a competitive binding FPA experiment.
[00147] For example, putative antagonist compounds (1 nM to 1 mM) and a fluoresceinated peptidomimetic macrocycle (25 nM) are incubated with the acceptor protein (50 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature. Antagonist binding activity ismeasured, for example, by fluorescence polarization on a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determined by nonlinear regression analysis using, for example, Graphpad Prism software (GraphPad Software, Inc., San Diego, CA).
[00148] Any class of molecule, such as small organic molecules, peptides, oligonucleotides or proteins can be examined as putative antagonists in this assay.
Assay for Protein-ligand binding by Affinity Selection-Mass Spectrometry
[00149] To assess the binding and affinity of test compounds for proteins, an affinity-selection mass spectrometry assay is used, for example. Protein-ligand binding experiments are conducted according to the following representative procedure outlined for a system- wide control experiment using 1 μΜ peptidomimetic macrocycle plus 5 μΜ target protein. A 1 iL DMSO aliquot of a 40 μΜ stock solution of peptidomimetic macrocycle is dissolved in 19 μΙ_, of PBS (Phosphate-buffered saline: 50 mM, pH 7.5 Phosphate buffer containing 150 mM NaCl). The resulting solution is mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To a 4 iL aliquot of the resulting supernatant is added 4 of 10 μΜ target protein in PBS. Each 8.0 μΐ^ experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μΜ concentration in PBS plus 1 μΜ peptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated for 60 min at room temperature, and then chilled to 4 °C prior to size-exclusion chromatography-LC-MS analysis of 5.0 injections. Samples containing a target protein, protein-ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from non-binding component by a rapid SEC step. The SEC column eluate is monitored using UV detectors to confirm that the early-eluting protein fraction, which elutes in the void volume of the SEC column, is well resolved from unbound components that are retained on the column. After the peak containing the protein and protein-ligand complexes elutes from the primary UV detector, it enters a sample loop where it is excised from the flow stream of the SEC stage and transferred directly to the LC-MS via a valving mechanism. The (M + 3H) ion of the peptidomimetic macrocycle is observed by ESI-MS at the expected m/z, confirming the detection of the protein-ligand complex.
Assay for Protein-ligand d Titration Experiments.
To assess the binding and affinity of test compounds for proteins, a protein-ligand Kd titration experiment is performed, for example. Protein-ligand Kd titrations experiments are conducted as follows: 2 μΕ DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (5, 2.5, 0.098 mM) are prepared then dissolved in 38 of PBS. The resulting solutions are mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To 4.0 μί aliquots of the resulting supernatants is added 4.0 μΐ^ of 10 μΜ hMDM2 in PBS. Each 8.0 μΐ^ experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μΜ concentration in PBS, varying concentrations (125, 62.5, 0.24 μΜ) of the titrant peptide, and 2.5% DMSO. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 30 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 μΕ injections. The (M + H)1+ , (M + 2H)2+ , (M + 3H)3+ , and/or (M + Na)1+ ion is observed by ESI-MS; extracted ion chromatograms are quantified, then fit to equations to derive the binding affinity as described in "A General Technique to Rank Protein-Ligand Binding Affinities and Determine AUosteric vs. Direct Binding Site Competition in Compound Mixtures ." Annis, D. A.; Nazef, N.; Chuang, C. C; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity Selection-Mass Spectrometry System or the Discovery and Characterization of Protein-Ligand Interactions" D. A. Annis, C- C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley- VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.
Assay for Competitive Binding Experiments by Affinity Selection-Mass Spectrometry
[00151] To determine the ability of test compounds to bind competitively to proteins, an affiinity selection mass spectrometry assay is performed, for example. A mixture of ligands at 40 μΜ per component is prepared by combining 2 μΕ aliquots of 400 μΜ stocks of each of the three compounds with 14 μΕ of DMSO. Then, 1 μΕ aliquots of this 40 μΜ per component mixture are combined with 1 μΕ DMSO aliquots of a serially diluted stock solution of titrant peptidomimetic macrocycle (10, 5, 2.5, 0.078 mM). These 2 μΕ samples are dissolved in 38 μΐ, of PBS. The resulting solutions were mixed by repeated pipetting and clarified by centrifugation at 10 OOOg for 10 min. To 4.0 μΐ^ aliquots of the resulting supernatants is added 4.0 μΐ^ of 10 μΜ hMDM2 protein in PBS. Each 8.0 μΐ^ experimental sample thus contains 40 pmol (1.5 μg) of protein at 5.0 μΜ concentration in PBS plus 0.5 μΜ ligand, 2.5% DMSO, and varying concentrations (125, 62.5, 0.98 μΜ) of the titrant peptidomimetic macrocycle. Duplicate samples thus prepared for each concentration point are incubated at room temperature for 60 min, then chilled to 4 °C prior to SEC-LC-MS analysis of 2.0 μί injections. Additional details on these and other methods are provided in "A General Technique to Rank Protein-Ligand Binding Affinities and Determine AUosteric vs. Direct Binding Site Competition in Compound Mixtures ." Annis, D. A.; Nazef, N.; Chuang, C. C; Scott, M. P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in "ALIS: An Affinity Selection-Mass Spectrometry System for the Discovery and Characterization of Protein-Ligand Interactions" D. A. Annis, C- C. Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G: Wiley- VCH; 2007:121-184. Mannhold R, Kubinyi H, Folkers G (Series Editors): Methods and Principles in Medicinal Chemistry.
Binding Assays in Intact Cells.
[00152] It is possible to measure binding of peptides or peptidomimetic macrocycles to their natural acceptors in intact cells by immunoprecipitation experiments. For example, intact cells are incubated with fluoresceinated (FITC-labeled) compounds for 4 hrs in the absence of serum, followed by serum replacement and further incubation that ranges from 4-18 hrs. Cells are then pelleted and incubated in lysis buffer (50mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and protease inhibitor cocktail) for 10 minutes at 4°C.
Extracts are centrifuged at 14,000 rpm for 15 minutes and supernatants collected and incubated with 10 μΐ goat anti-FITC antibody for 2 hrs, rotating at 4°C followed by further 2 hrs incubation at 4°C with protein A/G Sepharose (50 μΐ of 50% bead slurry). After quick centrifugation, the pellets are washed in lysis buffer containing increasing salt concentration (e.g., 150, 300, 500 mM). The beads are then re-equilibrated at 150 mM NaCl before addition of SDS-containing sample buffer and boiling. After centrifugation, the supernatants are optionally electrophoresed using 4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-P membranes. After blocking, blots are optionally incubated with an antibody that detects FITC and also with one or more antibodies that detect proteins that bind to the peptidomimetic macrocycle.
Cellular Penetrability Assays.
[00153] To measure the cell penetrability of peptidomimetic macrocycles and corresponding
uncrosslinked macrocycle, intact cells are incubated with fluoresceinated peptidomimetic macrocycles or corresponding uncrosslinked macrocycle (10 μΜ) for 4 hrs in serum free media at 37°C, washed twice with media and incubated with trypsin (0.25%) for 10 min at 37°C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics' KineticScan ® HCS Reader.
Cellular Antiviral Assays.
[00154] The efficacy of certain peptidomimetic macrocycles is determined, for example, in cell- based HIV infectivity assays using a variety of viral strains derived from human clinical populations. Cell viability is monitored, for example, over 24-96 hrs of incubation with peptidomimetic macrocycles (1 uM to 10 μΜ) to identify those that prevent HIV infection at EC50<10 μΜ. Several standard assays that measure cell viability are commercially available and are optionally used to assess the efficacy of the
peptidomimetic macrocycles.
In Vivo Stability Assay.
[00155] To investigate the in vivo stability of the peptidomimetic macrocycles, the compounds are, for example,administered to mice and/or rats by IV, IP, PO or inhalation routes at concentrations ranging from 0.1 to 50 mg/kg and blood specimens withdrawn at 0', 5', 15', 30', 1 hr, 4 hrs, 8 hrs and 24 hours post-injection. Levels of intact compound in 25 μΐ, of fresh serum are then measured by LC-MS/MS as above.
Clinical Trials. [00156] To determine the suitability of the peptidomimetic macrocycles of the invention for treatment of humans, clinical trials are performed. For example, patients diagnosed with HIV infection and in need of treatment are selected and separated in treatment and one or more control groups, wherein the treatment group is administered a peptidomimetic macrocycle of the invention, while the control groups receive a placebo or a known anti- HIV drug. The treatment safety and efficacy of the peptidomimetic macrocycles of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors such as survival and quality-of-life. In this example, the patient group treated with a peptidomimetic macrocyle show improved long-term survival compared to a patient control group treated with a placebo.
Pharmaceutical Compositions and Routes of Administration
[00157] The peptidomimetic macrocycles of the invention also include pharmaceutically
acceptable derivatives or prodrugs thereof. A "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention. Particularly favored pharmaceutically acceptable derivatives are those that increase the bioavailability of the compounds of the invention when administered to a mammal (e.g., by increasing absorption into the blood of an orally administered compound) or which increases delivery of the active compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Some pharmaceutically acceptable derivatives include a chemical group which increases aqueous solubility or active transport across the gastrointestinal mucosa.
[00158] In some embodiments, the peptidomimetic macrocycles of the invention are modified by covalently or non-covalently joining appropriate functional groups to enhance selective biological properties. Such modifications include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism, and alter rate of excretion.
[00159] Pharmaceutically acceptable salts of the compounds of this invention include those
derived from pharmaceutically acceptable inorganic and organic acids and bases.
Examples of suitable acid salts include acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4 + salts.
[00160] For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton PA.
[00161] In powders, the carrier is a finely divided solid, which is in a mixture with the finely
divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
[00162] Suitable solid excipients are carbohydrate or protein fillers include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl- cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents are added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
[00163] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
[00164] The pharmaceutical preparation is preferably in unit dosage form. In such form the
preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
[00165] When the compositions of this invention comprise a combination of a peptidomimetic macrocycle and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. In some embodiments, the additional agents are administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents are part of a single dosage form, mixed together with the compounds of this invention in a single composition. The amount of macrocycle that can be administered is not limited, and can range, for example, from about 2000 mg to about 1 mg, from about 1000 mg to about 1 mg, from about 500 mg to about 1 mg, or from about 100 mg to about 1 mg.
[00166] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Examples
Example 1 : Peptidomimetic macrocycles of the invention
[00167] Peptidomimetic macrocycles were synthesized, purified and analyzed as previously
described and as described below (Schafmeister et al, J. Am. Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem. Soc. 122:5891 (2005); Walensky et al, Science 305: 1466-1470 (2004); and US Patent No. 7,192,713). Peptidomimetic macrocycles were designed by replacing two or more naturally occurring amino acids with the corresponding synthetic amino acids. Substitutions were made at i and i+4, and i and i+7 positions. Peptide synthesis was performed either manually or on an automated peptide synthesizer (Applied Biosystems, model 433A), using solid phase conditions, rink amide AM resin (Novabiochem), and Fmoc main-chain protecting group chemistry. For the coupling of natural Fmoc-protected amino acids (Novabiochem), 10 equivalents of amino acid and a 1 : 1 :2 molar ratio of coupling reagents HBTU/HOBt
(Novabiochem)/DIEA were employed. Non-natural amino acids (4 equiv) were coupled with a 1 : 1 :2 molar ratio of HATU (Applied Biosystems)/HOBt/DIEA. The N-termini of the synthetic peptides were acetylated, while the C-termini were amidated.
[00168] Purification of cross-linked compounds was achieved by high performance liquid
chromatography (HPLC) (Varian ProStar) on a reverse phase CI 8 column (Varian) to yield the pure compounds. Chemical composition of the pure products was confirmed by LC/MS mass spectrometry (Micromass LCT interfaced with Agilent 1100 HPLC system) and amino acid analysis (Applied Biosystems, model 420A).
[00169] Tables 3 and 4 show a list of peptidomimetic macrocycles of the invention prepared.
Table 3
Figure imgf000070_0001
Figure imgf000070_0002
Table 4
Figure imgf000071_0001
[00170] In the sequences shown above and elsewhere, the following abbreviations are used: "Nle" represents norleucine, "Aib" represents 2-aminoisobutyric acid, "Ac" represents acetyl, and "Pr" represents propionyl. Amino acids represented as "$" are alpha-Me S5- pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond. Amino acids represented as "$r5" are alpha-Me R5- pentenyl-alanine olefin amino acids connected by an all-carbon i to i+4 crosslinker comprising one double bond. Amino acids represented as "$s8" are alpha-Me S8-octenyl- alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond. Amino acids represented as "$r8" are alpha-Me R8-octenyl-alanine olefin amino acids connected by an all-carbon i to i+7 crosslinker comprising one double bond. "Ahx" represents an aminocyclohexyl linker. The crosslinkers are linear all-carbon crosslinker comprising eight or eleven carbon atoms between the alpha carbons of each amino acid. Amino acids represented as "$/" are alpha-Me S5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "$/r5" are alpha-Me R5-pentenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "$/s8" are alpha-Me S8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "$/r8" are alpha-Me R8-octenyl-alanine olefin amino acids that are not connected by any crosslinker. Amino acids represented as "Amw" are alpha-Me tryptophan amino acids. Amino acids represented as "Ami" are alpha-Me leucine amino acids. Amino acids represented as "2ff ' are 2-fluoro-phenylalanine amino acids. Amino acids represented as "3ff ' are 3 -fluoro-phenyl alanine amino acids. Amino acids represented as "St" are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated. Amino acids represented as "St//" are amino acids comprising two pentenyl-alanine olefin side chains that are not crosslinked. Amino acids represented as "%St" are amino acids comprising two pentenyl-alanine olefin side chains, each of which is crosslinked to another amino acid as indicated via fully saturated hydrocarbon crosslinks.
Example 2: Metabolism by Purified Protease
[00171] Linear peptides and cross-linked peptidomimetic macrocycles were tested for stability to proteolysis by Trypsin (MP Biomedicals, Solon OH) by solubilizing each peptide at 10 μΜ concentration in 200 100 mM NH40Ac (pH 7.5). The reaction was initiated by adding 3.5 μΐ of Trypsin (12.5 μg protease per 500 μΐ, reaction) and shaking continually in sealed vials while incubating in a Room Temperature (22±2 <C). The enzyme/substrate ratio was 1 : 102 (w/w). After incubation times of 0, 5, 30, 60 and 135 min the reaction was stopped by addition of equal volume of 0.2% trifluoroacetic acid. Then, the solution was immediately analyzed by LC-MS in positive detection mode. The reaction half-life for each peptide was calculated in GraphPad Prism by a non-linear fit of uncalibrated MS response versus enzyme incubation time.
Example 3 : Anti-HIV Efficacy Evaluation in Fresh Human PBMCs
[00172] Fresh human PBMCs, seronegative for HIV and HBV, are isolated from screened donors (Biological Specialty Corporation, Colmar, PA). Cells are pelleted/washed 2-3 times by low speed centrifugation and re-suspension in PBS to remove contaminating platelets. The Leukophoresed blood is then diluted 1 : 1 with Dulbecco's Phosphate Buffered Saline (DPBS) and layered over 14 mL of Lymphocyte Separation Medium (LSM; Cellgro® by Mediatech, Inc.; density 1.078+/-0.002 g/ml; Cat.# 85-072-CL) in a 50 mL centrifuge tube and then centrifuged for 30 minutes at 600 X g. Banded PBMCs are gently aspirated from the resulting interface and subsequently washed 2X with PBS by low speed centrifugation. After the final wash, cells are enumerated by trypan blue exclusion and re-suspended at 1 x 107 cells/mL in RPMI 1640 supplemented with 15 % Fetal Bovine Serum (FBS), and 2 mM L-glutamine, 4 μg/mL Phytohemagglutinin (PHA, Sigma). The cells are allowed to incubate for 48-72 hours at 37°C. After incubation, PBMCs are centrifuged and re-suspended in RPMI 1640 with 15% FBS, 2 mM L- glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 20 U/mL recombinant human IL-2 (R&D Systems, Inc). IL-2 is included in the culture medium to maintain the cell division initiated by the PHA mitogenic stimulation. PBMCs are maintained in this medium at a concentration of 1-2 x 106 cells/mL with biweekly medium changes until used in the assay protocol. Cells are kept in culture for a maximum of two weeks before being deemed too old for use in assays and discarded. MDMs are depleted from the culture as the result of adherence to the tissue culture flask.
[00173] For the standard PBMC assay, PHA stimulated cells from at least two normal donors are pooled (mixed together), diluted in fresh medium to a final concentration of 1 x 106 cells/mL, and plated in the interior wells of a 96 well round bottom microplate at 50 μΕΛνεΙΙ (5 x 104 cells/well) in a standard format. Pooling (mixing) of mononuclear cells from more than one donor is used to minimize the variability observed between individual donors, which results from quantitative and qualitative differences in HIV infection and overall response to the PHA and IL-2 of primary lymphocyte populations. Each plate contains virus/cell control wells (cells plus virus), experimental wells (drug plus cells plus virus) and compound control wells (drug plus media without cells, necessary for MTS monitoring of cytotoxicity). In this in vitro assay, PBMC viability remains high throughout the duration of the incubation period. Therefore, infected wells are used in the assessment of both antiviral activity and cytotoxicity. Test drug dilutions are prepared at a 2X concentration in microtiter tubes and 100 of each concentration (nine total concentrations) are placed in appropriate wells using the standard format. 50 μΐ^ of a predetermined dilution of virus stock is placed in each test well (final MOI≥ 0.1). The PBMC cultures are maintained for seven days following infection at 37°C, 5% C02. After this period, cell-free supernatant samples are collected for analysis of reverse transcriptase activity and/or p24 antigen content. Following removal of supernatant samples, compound cytotoxicity is measured by addition of MTS to the plates for determination of cell viability. Wells are also examined microscopically and any abnormalities are noted.
[00174] A microtiter plate-based reverse transcriptase (RT) reaction is utilized (Buckheit et al., AIDS Research and Human Retroviruses 7:295-302, 1991). Tritiated thymidine triphosphate (3H-TTP, 80 Ci/mmol, NEN) is received in 1 : 1 dH20:Ethanol at 1 mCi/mL. Poly rA:oligo dT template :primer (Pharmacia) is prepared as a stock solution by combining 150 poly rA (20 mg/mL) with 0.5 mL oligo dT (20 units/mL) and 5.35 mL sterile dH20 followed by aliquoting (1.0 mL) and storage at -20°C. The RT reaction buffer is prepared fresh on a daily basis and consists of 125 1.0 M EGTA, 125 μΐ, dH20, 125 μΕ 20% Triton X100, 50 μΕ 1.0 M Tris (pH 7.4), 50 μΕ 1.0 M DTT, and 40 μΕ 1.0 M MgC12. The final reaction mixture is prepared by combining 1 part 3H-TTP, 4 parts dH20, 2.5 parts poly rA:oligo dT stock and 2.5 parts reaction buffer. Ten microliters of this reaction mixture is placed in a round bottom microtiter plate and 15 μί of virus containing supernatant is added and mixed. The plate is incubated at 37°C for 60 minutes. Following incubation, the reaction volume is spotted onto DE81 filter-mats (Wallac), washed 5 times for 5 minutes each in a 5% sodium phosphate buffer or 2X SSC (Life Technologies). Next they are washed 2 times for 1 minute each in distilled water, 2 times for 1 minute each in 70% ethanol, and then dried. Incorporated radioactivity (counts per minute, CPM) is quantified using standard liquid scintillation techniques.
Example 4: Anti-HIV Efficacy Evaluation in Fresh Human MDMs [00175] Peripheral blood monocytes are isolated from screened donors, seronegative for HIV and HBV. Cells are pelleted/washed 2-3 times by low speed centrifugation and re-suspension in PBS to remove contaminating platelets. The Leukophoresed blood is then diluted 1 : 1 with Dulbecco's phosphate buffered saline (PBS) and layered over 14 mL of
Lymphocyte Separation Medium (LSM; cellgro® by Mediatech, Inc.; density 1.078+/- 0.002 g/ml; Cat.# 85-072-CL) in a 50 mL centrifuge tube and then centrifuged for 30 minutes at 600 X g. Banded PBMCs are gently aspirated from the interface and subsequently washed 2X with PBS by low speed centrifugation. The cells are diluted to 4 x 106 cells per mL in DMEM without phenol red supplemented with 10% heat inactivated human pooled AB serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Monocytes-derived-macrophages (MDMs) are allowed to adhere to the interior 60 wells (100 μίΛνεΙΙ) of a 96 well flat bottomed plate for 2 to 18 hours at 37°C, 5% C02. The exterior wells are filled with 200 of sterile DPBS to serve as a humidity barrier. Following adherence, the cultures are washed with sterile DPBS to remove non-adherent cells (lymphocytes and contaminating RBCs). 200 L of RPMI 1640 supplemented with 15 % FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin is subsequently added to the wells. The plates are incubated at 37°C in a humidified incubator with 5% C02. Culture medium is replaced once per week until use. Culture plates are used for anti-HIV evaluations between days 6 and 14 of incubation following initial isolation of the cells.
[00176] Following 6 to 14 days in culture, the MDM cultures are washed 3 times to remove any non-adherent cells and serially diluted test compounds are added followed by the addition of a pre-titered amount of HIV. Cultures are washed a final time by media removal 24 hours post infection, fresh compound added and the cultures continued for an additional six days. The assays are performed using a standardized microtiter plate format. Each plate contains virus/cell control wells (cells plus virus), experimental wells (drug plus cells plus virus) and compound control wells (drug plus media without cells, necessary for MTS monitoring of cytotoxicity). In this in vitro assay, macrophage viability remains high throughout the duration of the incubation period. Therefore, infected wells are used in the assessment of both antiviral activity and cytotoxicity. At assay termination, virus replication is measured by collecting cell-free supernatant samples, which are analyzed for HIV-1 p24 antigen content using a commercially available p24 ELISA assay (e.g., PerkinElmer). Following removal of supernatant samples, compound cytotoxicity is measured by addition of MTS to the plates for determination of cell viability. Wells are also examined microscopically and any abnormalities noted.
P24 antigen ELISA kits are purchased from PerkinElmer or other similar vendors. The assay is performed according to the manufacturer's instructions. Control curves are generated in each assay to accurately quantify the amount of p24 antigen in each sample. Data are obtained by spectrophotometric analysis at 490/670 nm using a Molecular Devices SpectraMaxPlus plate reader. Final concentrations are calculated from the optical density values using the Molecular Devices SOFTmax Pro software package.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1, 2, 3, and 4, wherein the peptidomimetic macrocycle has an IC50 of about 10000 nM or less against a target and in cells selected from the group consisting of:
target: HIV-1 IIIB, cells: CEM-SS; target: HIV-1NL4-3, cells: MT-4; and target: HIV- 1MDR769, cells: hPBMC; wherein the peptidomimetic macrocycle is not a compound having the sequence:
(SP-13)YT$LI H$LIEESQNQQEKNEQELLEL$KW A $ L W NWF,
(SP-33) MeTWSEWD $EINNYTSLIHSL IEESQN$QEK$EQE L L E, and
SP-41)NleTW$EWD$EINNYT$LIH$LI EESQNQ$E KN$ Q ELLE.
2. The peptidomimetic macrocycle of claim 1, wherein the amino acid sequence of the peptidomimetic macrocycle is at least about 80% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1, 2, 3, and 4.
3. The peptidomimetic macrocycle of claim 1, wherein the amino acid sequence of the peptidomimetic macrocycle is at least about 90% identical to an amino acid sequence selected from the group consisting of the amino acid sequences in Tables 1, 2, 3, and 4.
4. The peptidomimetic macrocycle of claim 1, wherein the amino acid sequence of the peptidomimetic macrocycle is selected from the group consisting of the amino acid sequences in Tables 1, 2, 3, and 4.
5. The peptidomimetic macrocycle any of claims 1-4, having a positive number of amino acids of 60 or less.
6. The peptidomimetic macrocycle of any of claims 1-4, having a positive number of amino acids of 50 or less.
7. The peptidomimetic macrocycle of any of claims 1-4, having a positive number of amino acids of 40 or less.
8. The peptidomimetic macrocycle of any preceeding claim, comprising one or more independent crosslmkers, each crosslmker independently linking the a-positions of two amino acids of the peptidomimetic macrocycle.
9. The peptidomimetic macrocycle of claim 8, wherein at least one of the two amino acids is an α,α-disubstituted amino acid.
10. The peptidomimetic macrocycle of any of claims 1-9, comprising one crosslinker linking the a-positions of two amino acids of the peptidomimetic macrocycle.
11. The peptidomimetic macrocycle of any of claims 1-9, comprising two
independent crosslinkers, each crosslinker independently linking the a-positions of two amino acids of the peptidomimetic macrocycle.
12. A peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to HIV gp41 626-663, further comprising at least two independent crosslinkers, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid,
wherein the first amino acid and the second amino acid are selected from the group consisting of amino acids: 629 and 633; 630 and 634; 632 and 636; and 633 and 637; wherein the third amino acid and the fourth amino acid are selected from the group consisting of amino acids: 650 and 654; 651 and 655; 652 and 656; 653 and 657; 654 and 658; 655 and 659; 656 and 660; and 657 and 771;
with the proviso that when the first crosslinker connects amino acids pairs 629 and 633 and the second crosslinker connects amino acid pair 653 and 657, there is an additional crosslinker that connects amino acids pairs 641 and 645 or 642 and 646.
13. The peptidomimetic macrocycle of claim 12, comprising the additional cross linker, wherein the additional crosslinker connects an amino acid pair selected from the group consisting of: 640 and 644; 641 and 645; and 642 and 646.
14. The peptidomimetic macrocycle of claim 12, wherein the first crosslinker connects amino acid pair 629 and 633, and the second crosslinker connects amino acid pair 654 and 658.
15. The peptidomimetic macrocycle of claim 14, having a sequence that is at least about 60% identical to the sequence:
Nle T W $ E W D $ E I N N Y T S L I H S L I E E S Q N Q Q $ K N E $ E L L E
(SP-36).
16. The peptidomimetic macrocycle of claim 12, wherein the first crosslinker connects amino acid pair 629 and 633, and the second crosslinker connects amino acid pair 655 and 659.
17. The peptidomimetic macrocycle of claim 16, having a sequence that is at least about 60%) identical to the sequence: Nle TW$EWD$EINNYTSLIHSLIEESQNQQE$NEQ$LLE
(SP-37).
18. The peptidomimetic macrocycle of claim 12, wherein the first crosslinker connects amino acid pair 633 and 637, and the second crosslinker connects amino acid pair 655 and 659.
19. The peptidomimetic macrocycle of claim 18, having a sequence that is at least about 60% identical to the sequence:
Nle T W Nle EWD$EIN$YTSLIHSLIEESQNQQE$NEQ$LL
E (SP-48).
20. A peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60% identical to HIV gp41638-673, further having one crosslinker, wherein the crosslinker connects a first amino acid to a second amino acid, wherein the first amino acid and the second amino acid are 652 and 659.
21. A peptidomimetic macrocycle comprising an amino acid sequence which is at least about 60%> identical to HIV gp41638-673, further comprising at least two independent crosslinkers connecting at least two pairs of amino acids, wherein the first crosslinker connects a first amino acid to a second amino acid, and the second crosslinker connects a third amino acid to a fourth amino acid, wherein each pair of first and second amino acids are selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671;
wherein when the first crosslinker connects amino acid pair 640 and 644, the second crosslinker does not connect the amino acid pair 664 and 668 or 666 and 670; and
wherein when the first crosslinker connects amino acid pair 653 and 657, the second crosslinker does not connect amino acid pair 666 and 670.
22. The peptidomimetic macrocycle of claim 21, wherein the first crosslinker connects an amino acid pair selected from the group consisting of: 640 and 644; 641 and 645; 642 and 646; 646 and 650; 652 and 656; 652 and 659; and 653 and 657.
23. The peptidomimetic macrocycle of claim 21, wherein the second crosslinker connects an amino acid pair selected from the group consisting of: 652 and 656; 652 and 659; 653 and 657; 661 and 665; 661 and 668; 662 and 666; 662 and 669; 664 and 668; 664 and 671; 665 and 669; 666 and 670; and 667 and 671.
24. The peptidomimetic macrocycle of any of claims 12-23, comprising a helix.
25. The peptidomimetic macrocycle of claim 24, wherein the helix is an a-helix.
26. The peptidomimetic macrocycle of any of claims 12-25, comprising an α,α- disubstituted amino acid.
27. The peptidomimetic macrocycle of any of claims 12-26, wherein at least one of the two amino acids in one or more amino acid pairs is an α,α-disubstituted amino acid.
28. The peptidomimetic macrocycle of any preceding claim, having the Formula (I):
Figure imgf000080_0001
Formula I
wherein:
each A, C, D, and E is independently a n
B is a natural or non-natural amino acid,
Figure imgf000080_0002
[-NH-L3-
CO-], [-NH-L3-SO2-], or [-NH-L3-];
Ri and R2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is unsubstituted or substituted with halo-;
R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which except hydrogen is optionally substituted with R5;
L is a macrocycle-forming linker of the formula -Li-L2-;
Li and L2 and L3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R4-]n, each being optionally substituted with R5;
each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene;
each K is O, S, SO, S02, CO, C02, or CONR3; each R5 is independently halogen, alkyl, -OR6, -N(R6)2, -SR6, -SOR5, -S02R6, -C02R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
each R6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
R7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R5, or part of a cyclic structure with a D residue;
R8 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R5, or part of a cyclic structure with an E residue;
v and w are independently integers from 1-1000, for example: 1-500, 1-300, 1-200, 1- 100, 1-50, 1-40, 1-30, 1-20, 1-10;
u is an integer from 1-10, for example 1, 2, or 3;
x, y and z are independently integers from 0-10; for example, the sum of x+y+z can equal 2, 3 or 6, for example, 3 or 6; and
n is an integer from 1-5.
29. The peptidomimetic macrocycle of claim 28, wherein Li and L2 are independently alkylene, alkenylene or alkynylene.
30. The peptidomimetic macrocycle of claim 28, wherein Li and L2 are independently C3-C10 alkylene or alkenylene
31. The peptidomimetic macrocycle of claim 28, wherein Li and L2 are independently C3-C6 alkylene or alkenylene.
32. The peptidomimetic macrocycle of claim 28, wherein Ri and R2 are H.
33. The peptidomimetic macrocycle of claim 28, wherein Ri and R2 are
independently alkyl.
34. The peptidomimetic macrocycle of claim 28, wherein Ri and R2 are methyl.
35. The peptidomimetic macrocycle of any of claims 1-27, having the Formula (II) or
(Ila):
Figure imgf000081_0001
Figure imgf000082_0001
— ' u Formula (Ila), wherein:
each A, C, D, and E is independently a natural or non-natural amino acid;
R3
B is a natural or non-natural amino acid, amino acid analog, O , [-NH-L3-CO-],
[-NH-L3-SO2-], or [-NH-L3-];
Ri and R2 are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, each of which except -H is unsubstituted or substituted with halo-, or part of a cyclic structure with an E residue;
R3 is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, each of which except hydrogen is optionally substituted with R5;
Li and L2 and L3 are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R4-]n, each being optionally substituted with R5;
each R4 is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene;
each K is O, S, SO, S02, CO, C02, or CONR3;
each R5 is independently halogen, alkyl, -OR6, -N(R6)2, -SR6, -SORs, -S02R6, -C02R6, a fluorescent moiety, a radioisotope or a therapeutic agent;
each R6 is independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent;
R7 is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, each of which except -H is optionally substituted with R5;
v and w are independently integers from 1-1000;
u is an integer from 1-10, for example 1, 2, or 3; x, y and z are independently integers from 0-10; for example, the sum of x+y+z can equal 2, 3 or 6, for example, 3 or 6; and
n is an integer from 1-5.
36. The peptidomimetic macrocycle of any of claims 12, 14-19, and 21-27, having the Formula (III):
Figure imgf000083_0001
wherein each A, C, D, and E is independently an amino acid;
R3
B is an amino acid, O , [-NH-L3-CO-], [-NH-L3-SO2-], or [-NH-L3-];
L' is a macrocycle-forming linker of the formula -Li'-L2'-;
and wherein A, B, C, D, and E, taken together with the crosslinked amino acids connected by the macrocycle-forming linkers L and L', form the amino acid sequence of the peptidomimetic macrocycle;
Ri' and R2' are independently -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-;
Li ' and L2' are independently alkylene, alkenylene, alkynyl ene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [-R4-K-R4-]n, each being optionally substituted with R5;
each K is independently O, S, SO, S02, CO, C02, or CONR3;
R7' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with a D residue;
Rs' is -H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R5, or part of a cyclic structure with an E residue;
v' and w' are independently integers from 1-100;
x', y' and z' are independently integers from 0-10; and
n is an integer from 1-5.
37. A method of preventing HIV infection in a subject comprising administering to the subject a peptidomimetic macrocycle of any of claims 1-36.
38. A method of treating HIV infection and associated immunodeficiencies in a subject comprising administering to the subject a peptidomimetic macrocycle of any of claims 1- 36.
39. A method of inhibiting HIV viral entry in a subject comprising administering to the subject a peptidomimetic macrocycle of any of claims 1-36.
40. A method of antagonizing the formation of the six -helix bundle HIV HIV gp41 fusion complex in a subject comprising administering to the subject a peptidomimetic macrocycle of any of claims 1-36.
41. A method of preventing or treating HIV infection and associated
immunodeficiencies (i.e., AIDS) in a subject comprising administering an antagonist of the six- helix bundle HIV HIV gp41 fusion complex, wherein the antagonist is administered no more frequently than once daily, no more frequently than every other day, no more frequently than twice weekly, no more frequently than weekly, or no more frequently than every other week, and wherein the antagonist is a peptidomimetic macrocycle of any of claims 1-36.
42. A method of preventing or treating HIV infection and associated
immunodeficiencies (i.e., AIDS) in a subject comprising administering an antagonist of the six- helix bundle HIV HIV gp41 fusion complex, wherein the frequency or intensity of the site of injection reaction is reduced relative to that of enfuvirtide, wherein the antagonist is a peptidomimetic macrocycle of any of claims 1-36.
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