WO2014036198A1 - Compositions and methods for specific inhibition of leishmania receptor for activated c-kinase (lack) - Google Patents
Compositions and methods for specific inhibition of leishmania receptor for activated c-kinase (lack) Download PDFInfo
- Publication number
- WO2014036198A1 WO2014036198A1 PCT/US2013/057175 US2013057175W WO2014036198A1 WO 2014036198 A1 WO2014036198 A1 WO 2014036198A1 US 2013057175 W US2013057175 W US 2013057175W WO 2014036198 A1 WO2014036198 A1 WO 2014036198A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- peptide
- peptides
- protein
- modulatory
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This disclosure relates generally to rationally designed anti-parasitic peptide inhibitors of receptor for activated C-kinase (RACK) orthologs, known in Leishmania species as “LACK” and in Trypanosoma cruzi (T cruzi) as “TRACK,” and methods of their use for protection against infection by parasites, as well as for inhibition and treatment for neglected tropical diseases, such as, for example Leishmaniasis and Chagas Disease.
- RACK receptor for activated C-kinase
- NTDs Neglected tropical diseases
- leishmaniasis and trypanosomiasis affect millions of people mainly in poor countries and rural areas throughout the Americas.
- Trypanosoma cruzi is the causative agent of Chagas disease, a potentially life-threatening illness which is endemic in South and Central America, affecting approximately 12 million people in Latin America, resulting in an incidence of 21 ,000 deaths (Teixeira, 2006).
- Chagas disease is mostly transmitted to humans by the feces of triatominae bugs, sometimes known as 'kissing bugs,' depending on the geographical area.
- NTD complicity can be related to the sophisticated defense mechanisms that parasites have developed against mammalian immune system, demonstrated partially by lipophosphoglycans that are found on the parasite surface and used by the parasite to promote its survival in the host (Hutchinson, 2007; Barrett, 2007).
- most treatments for neglected tropical diseases are prohibitively expensive, typically require multiple doses by injection and commonly have unacceptable toxicity. Frequently, drug resistance also arises (Barrett, 2003; Bouteille, 2003; Bray, 2003).
- Trypanosomatids are protozoan parasites distinguished by having only a single flagellum. The name is derived from the Greek trypano (borer) and soma (body), indicative of the corkscrew-like motion of some trypanosomatid species. Trypanosomatid parasites are found primarily in insects; however, a few genera have life-cycles involving a secondary host, which may be a vertebrate, invertebrate or plant.
- Leishmania is a genus of Trypanosomatid protozoa, and is the parasite responsible for the disease leishmaniasis.
- the disease is transmitted by the bite of female sandflies of the genera Phlebotomus in the Old World and Lutzomyia in the New World.
- New World Lutzomyia sandflies transmit the Leishmania amazonensis parasite
- Old World Phlebotomus transmit Leishmania donovani, whose primary hosts are vertebrates.
- Leishmania commonly infects hyraxes, canids, rodents, and humans.
- Leishmaniasis is the second-largest parasitic killer in the world, and currently affects 12 million people in tropical and subtropical regions of over 80 countries, with 1.5 to 2 million new cases occurring annually, and 59,000 deaths occurring in 2001 alone (See who.int/tdr/diseases/leish/diseaseinfo.htm). To date, no vaccines are commercially available for leishmaniasis and the current treatment is toxic; furthermore, drug resistance to the most commonly used medication has been reported (Singh, 2003).
- Described herein is an alternative approach to rational drug design, which focuses on modifying protein-protein interactions in parasites.
- Effective signal transduction pathways rely on correct distribution of regulatory proteins within the cell.
- Anchor proteins have been described to serve as scaffolds upon which signal complexes can assemble, helping to provide spatial organization to signal transduction processes by allowing the formation of multimeric complexes at appropriate locations in the cell.
- Association with different anchors allows some signal kinases to participate in and distinguish between multiple pathways.
- PKC Protein kinase C
- lipid-derived second messengers and in some cases, calcium
- Various activated PKC isoforms interact with various Receptors for Activated C-Kinase (“RACKs").
- the Receptor for Activated C-Kinase 1 (RACK1 ) is a ubiquitous and highly conserved scaffold protein having WD repeats, and it acts an anchoring protein of ⁇ , helping to regulate a range of cell activities including cell growth, survival, differentiation, shape and protein translation by recruiting signal proteins to specific nuclear or plasma membrane sites, to the cytoskeleton or to the 40S ribosome.
- RACK1 forms productive ternary complexes with a wide range of signal protein partners; target proteins interact with RACK1 through SH2 domains, PH domains, C2 domains, and other specific sequences.
- Peptides representing these unique sequences have been found to act as competitive inhibitors, disrupting the protein-protein interaction between the PKC isozyme and its corresponding RACK, thereby inhibiting the functions of a given PKC isozyme.
- inhibitory intra-molecular protein-protein interactions have been observed to keep the enzyme in the inactive state.
- At least one such intra-molecular interaction has been identified between the RACK-binding site in PKC and a sequence in the inactive form of a PKC isozyme which has amino acid sequence similarity to a site in the corresponding RACK; thus, the inactive PKC occludes the RACK binding site with a peptide sequence known as the pseudo-RACK ⁇ RACK) (Dorn et al., 1999, Proc. Natl., Acad. Sci., 96:12798-12803).
- a peptide corresponding to this l + J RACK site competes with the intra-molecular inhibitory interaction, thus serving as a selective activator of the corresponding isozyme.
- RACK1 is ubiquitous and highly conserved scaffold protein found in various species, including plants, parasites and yeast (Enserink, 2000; McCahill, 2002). RACK1 has been shown to be involved in many signaling processes leading to cell survival, growth and differentiation (reviewed in Schechtman, 2001 ). In Schizosaccharomyces pombe, the RACK1 homolog (Cpc2) associates with the ribosome, is essential for yeast metabolism. (Hoffmann, 1999) growth regulation, differentiation, and cell cycle progression. Yeast that do not express Cpc2 are viable but have a longer cell cycle (Shor, 2003; McLeod, 2000).
- Cpc2 is a scaffold protein that binds and regulates the activity of key signaling molecules such as a PKC yeast homolog Pck2 (Won, 2001 ) and Rani (Pat1 ) kinase (involved in cell cycle progression) (McLeod, 2000).
- key signaling molecules such as a PKC yeast homolog Pck2 (Won, 2001 ) and Rani (Pat1 ) kinase (involved in cell cycle progression) (McLeod, 2000).
- LACK Leishmania homologue of receptors for activated C kinase, RACK; also known as Leishmania activated C kinase receptor homologue
- RACK Leishmania homologue of receptors for activated C kinase
- LACK is found in both the amastigotes and promastigotes, and because of its homology to RACK, LACK is also believed to be a scaffolding protein, interacting with multiple signaling enzymes in these parasites, involved in essential signaling processes.
- LACK In Leishmania major, there are four LACK genes encoding identical proteins, which appear to play an important role in the life of the parasite, as genetic knockouts of LACK are not viable (Locksley et al.), and parasites containing a single copy of the gene and expressing low levels of LACK fail to parasitize even immunocompromised mice (Kelly, 2003). LACK is found in a multiprotein complex in the parasite kinetoplast, likely bound to signaling enzymes involved in DNA replication (Gonzalez-Aseguinolaza, 1999). In Trypanosoma brucei (T.
- TbRACKI the RACK1 homologue
- TRACK Trypanosome activated C kinase receptor homologue
- TbRACKI is a component of the translation machinery.
- RNA interference was used to inhibit TbRACKI , initiation of translation and phosphorylation of a ribosomal protein were inhibited (Regmi, 2008).
- TRACK is required for cytokinesis, and knockdown of TRACK impaired parasite growth (Rothberg, 2006). Infected mice with depleted TbRACKI caused elimination of circulating blood forms.
- LACK and TRACK have been shown to be essential for parasite survival/infectivity; thus, in the course of the present disclosure, LACK and TRACK were selected as drug targets, and certain regions of these parasite scaffold proteins involved in protein-protein interactions were identified and peptides derived from these regions were designed.
- LACK and TRACK were selected as drug targets, and certain regions of these parasite scaffold proteins involved in protein-protein interactions were identified and peptides derived from these regions were designed.
- LACK-BP LACK-binding protein
- amazonensis promastigotes parasite viability and inducing parasite cell death (IC5 0 ⁇ 10 ⁇ )), as well as the parasite's ability to infect macrophages, while the peptides themselves were found to be non-toxic to macrophages.
- These peptide inhibitors provide the basis for prophylactic and/or therapeutic compositions for protection against infection by Leishmania species, as well as providing useful leads in the development of novel inhibitors for treatment for neglected tropical diseases (NTD) such as Cutaneous Leishmaniasis, Mucosal Leishmaniasis, Visceral Leishmaniasis and Chagas disease.
- NTD neglected tropical diseases
- the novel peptides of the present disclosure are effective against a variety of Leishmania species from both Old and New worlds.
- the methods described herein are very likely applicable in the rational design of other anti-parasitic drugs.
- a peptide that selectively modulates the binding of a LACK or TRACK protein to its cognate binding protein comprises a contiguous sequence of 5 amino acid residues, wherein said contiguous sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ I D NO: 10, SEQ ID NO:1 1 , SEQ I D N0: 12, SEQ ID N0:13, SEQ ID N0:14 SEQ ID NO: 15, SEQ I D NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ I D NO: 19, SEQ I D NO: 20, SEQ ID NO: 21 , SEQ I D NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ I D NO: 25, SEQ I D NO: 26, SEQ ID NO: 27, SEQ I D NO:
- the modulatory peptide consists of 5-20 amino acid residues.
- the modulatory peptide is selected from the group consisting of SEQ I D NO: 3, SEQ I D NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ I D NO: 8, SEQ I D NO: 9, SEQ ID NO: 10, SEQ I D NO: 1 1 , SEQ I D NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ I D NO: 21 , SEQ I D NO: 22, SEQ I D NO: 23, SEQ I D NO: 24, SEQ I D NO: 25, SEQ ID NO: 26, SEQ I D NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ I D NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ I D NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
- a selective modulatory composition comprising the peptide.
- the composition further comprises a pharmaceutical
- a modulatory peptide (also known as a "cargo" peptide) is provided, wherein the peptide is linked to a carrier peptide sequence to enable transmembranal delivery.
- the carrier peptide is a TAT peptide based on the Tat protein of Human Immunodeficiency Virus (HIV).
- the carrier peptide is TAT 47- 57.
- the carrier peptide is at least 60% identical to the sequence RRRQRRKKRGY (herein set forth as SEQ ID NO:1 ).
- the carrier peptide is the sequence RRRQRRKKRGY (SEQ I D NO:1 ).
- the amino acid sequence of the TAT peptide is reversed with respect to the amino and carboxy termini of SEQ ID NO: 1 or SEQ ID NO: 2.
- the carrier peptide is at least 60% identical to the sequence YGRKKRRQRRR based on the Tat protein of Human Immunodeficiency Virus (HIV) (SEQ ID NO:2).
- the carrier peptide is the sequence YGRKKRRQRRR (SEQ ID NO:2).
- the modulatory peptide is linked to a spacer.
- the spacer is a glycine-glycine dipeptide ("GG”).
- the spacer is a glycine-serine-glycine tripeptide ("GSG”).
- Other linkers are a beta-Alanine (“ ⁇ -Ala” or “b-Ala"), cysteine (Cys), gamma-amino-n-butyric acid (also known as "GABA” or “Gaba”), or Fmoc-Epsilon-Ahx-OH.
- the modulatory peptide is not linked to a spacer.
- the modulatory peptide is linked to a cargo peptide, directly or indirectly. In some embodiments, the modulatory peptide is linked to a spacer which is linked to a cargo peptide.
- the carrier sequences e.g., SEQ ID NO: 1 or SEQ ID NO: 2 are used as the cargo component. In some embodiments, a cargo sequence (e.g., SEQ ID NO: 3) is used as a carrier component.
- the carrier peptide further comprises a sulfur-containing residue, which can be useful for conjugation. In some embodiments, the sulfur-containing residue is cysteine. In some embodiments, the sulfur-containing residue is a cysteine analog.
- the carrier peptide further comprises a cysteine residue attached via a peptide bond to its C-terminus or N- terminus.
- a cysteine residue is present as an alternative residue at any position within the carrier peptide or the cargo peptide.
- the modulatory peptide comprises of 5-20 amino acid residues. In another embodiment, the modulatory peptide comprises of 5-15 amino acids, 5-10 amino acids, 6-15 amino acids, 6-10 amino acids, or 6-8 amino acids. In still another embodiment, the modulatory peptide comprises 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 1 1 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 amino acids.
- the modulatory peptide is selected from the group consisting of CVSLAHATD (SEQ ID NO: 3), RNGQCQRK (SEQ ID NO: 5), MHEFLRD (SEQ ID NO: 6), NVIRVWN (SEQ ID NO: 7), VNGGKCERTLK (SEQ ID NO: 8), STGEQLFKINVESP (SEQ ID NO: 9), TPDGAKPSE (SEQ ID NO:10), RSLSVYD (SEQ ID NO: 1 1 ), QKKGDITDPYVKL (SEQ ID NO: 12), KTSVVRNN (SEQ ID NO: 13), GLNPYWMET (SEQ ID NO: 14), (QQAGSYIKVV) SEQ ID NO: 21 , (RHSVDSDYGLPSH) SEQ ID NO: 22, (EGHLKGHRGW) SEQ ID NO: 23, (DGTAIS) SEQ ID NO: 24, (ANPDRHSVS) SEQ ID NO: 25, (GLPSHRLE) S
- the modulatory peptide is not (SEQ ID NO: 3), (SEQ ID NO: 5), (SEQ ID NO: 6), (SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID NO:10), (SEQ ID NO: 1 1 ), (SEQ ID NO: 12), (SEQ ID NO: 13), (SEQ ID NO: 14), SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
- the modulatory peptide is at least about 60% identical to a contiguous sequence of equal length of a peptide sequence occurring in or derived from a substrate or cargo protein such as, for example (but not limited to) calmodulin A from Trypanosoma brucei (T. brucei) (herein set forth as SEQ ID NO: 15), calmodulin (e.g., SEQ ID NO: 16), heat shock protein from T. cruzi (herein set forth as SEQ ID NO: 17), elongation factor from T. cruzi (herein set forth as SEQ ID NO: 18) or heat shock protein 70 from T. cruzi (herein set forth as SEQ ID NO: 19).
- the modulatory peptide is at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a contiguous sequence of equal length derived from said protein or peptide.
- the modulatory peptide acts indirectly to inhibit activity of a downstream effector of a Protein Kinase C (PKC) isoform.
- PKC Protein Kinase C
- the modulatory peptide further comprises a sulfur-containing residue.
- the sulfur-containing residue is cysteine.
- the sulfur-containing residue is a cysteine analog.
- the sulfur-containing residue is located at the N-terminus and/or the C-terminus of the modulatory peptide. In another embodiment, the sulfur- containing residue is an internal residue.
- the modulatory peptide is linked to a carrier peptide. In one embodiment, the modulatory peptide is linked to a carrier peptide by a disulfide bond. In another embodiment, the modulatory peptide is linked to the carrier peptide by a peptide bond, wherein the modulatory peptide and the carrier form a single modulatory fusion peptide.
- a method for treating a subject comprising administration of a peptide or composition that selectively modulates the binding of a LACK or TRACK to its cognate binding partner.
- the subject is suffering from cutaneous leishmaniasis, mucosal leishmaniasis, visceral leishmaniasis, Chagas disease and/or trypanosomiasis.
- the method comprises administering the modulatory peptide to a subject in need thereof.
- the subject is undergoing or has undergone treatment with another antiparasitic agent.
- a method for reducing parasite infectivity comprising administering to a subject in need thereof, a peptide that selectively modulates the binding of a LACK or TRACK to its cognate binding partner.
- a method for modulating the activity of effectors downstream of a PKC is provided.
- Figures 1A and 1 B illustrate exemplary modulatory peptides comprising SEQ ID NO: 3.
- Figure 2 shows exemplary modulatory peptides comprising SEQ ID NO: 5.
- Figure 3 shows exemplary modulatory peptides comprising SEQ ID NO: 10.
- Figure 4 illustrates an exemplary in vivo peptide study protocol.
- Figures 5-9 present the results of in vivo experiments with several exemplary modulatory peptides.
- Figure 10 summarizes the effects of a modulatory peptide on parasite viability in a mouse model system.
- the carrier peptide comprises an amino acid sequence identified by SEQ ID NO: 1.
- the carrier peptide comprises an amino acid sequence identified by SEQ ID NO: 2.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 3.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 4.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 5.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 6.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 7. [0046] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 8.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 9.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 10.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 1 1.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 12.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 13.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 14.
- the LACK- or TRACK-binding protein is SEQ ID NO: 15.
- the LACK- or TRACK-binding protein is SEQ ID NO: 16.
- the LACK- or TRACK-binding protein is SEQ ID NO: 17.
- the LACK- or TRACK-binding protein is SEQ ID NO: 18.
- the LACK- or TRACK-binding protein is SEQ ID NO: 19.
- the LACK- or TRACK-binding protein is SEQ ID NO: 20.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 21.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 22.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 23.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 24.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 25. [0064] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 26.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 27.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 28.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 29.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 30.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 31.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 32.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 33.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 34.
- the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 35.
- the Leishmania parasite life cycle includes two forms: non-motile amastigotes and flagellate promastigote forms.
- a vector e.g. sandfly
- amastigotes are released into the vector ' s stomach.
- the parasites transform into promastigote forms inside the vector, and multiply in the extracellular blood and tissue fluids.
- the infected vector next feeds on a host by piercing the skin using his proboscis and injecting saliva which also contains anti-coagulant to avoid blood clotting, and the parasite.
- promastigotes infect the host, they are taken up by macrophages and the parasites revert from promastigotes to amastigote forms and the cycle continues.
- the present disclosure provides a different approach aimed at interfering with protein-protein interactions between the scaffold proteins homologous to RACK1 and their cognate binding partners, i.e., the Leishmania homologue Leishmania receptor for activated C kinase (LACK) and the Trypanosoma homologue, Trypanosoma receptor for activated C kinase (TRACK) with their binding partners.
- LACK Leishmania homologue Leishmania receptor for activated C kinase
- TRACK Trypanosoma receptor for activated C kinase
- LACK-deficient parasites are not viable (Kelly, 2003), and parasites expressing lower levels of LACK fail to parasitize even immune compromised-mice. LACK also scaffolds a multi-protein complex in the parasite (Gonzalez-Aseguinolaza, 1999) and it is involved in parasite signaling processes and protein synthesis (Choudhury, 201 1 ).
- TRACK Trypanosoma brucei
- RNAi RNA-binding protein
- TRACK and LACK are conserved amongst different Trypanosoma and Leishmania species, and there is high homology between parasitic species, while there is less homology between TRACK or LACK and mammalian RACK1 , suggesting that certain peptide inhibitors of protein-protein interactions could be effective against both Trypanosome and Leishmania species while not affecting mammalian host proteins.
- the rationally designed peptides developed herein were based on the homology and structure of LACK and TRACK to RACK1 , and were found to bind a previously described TRACK binding protein, elF1A, as well as to dramatically decrease the viability and infectivity of parasites.
- a structure activity relationship study based on these lead compounds allowed the identification of those exhibiting an increase in peptide activity against the parasite, reducing the number of viable free forms and intracellular parasites.
- the approach previously used to inhibit PKC-scaffold protein interaction and consequently PKC signaling was also effective in inhibiting the interaction of this parasite scaffold protein with its binding partners, resulting in parasite death.
- the modulatory peptides described herein represent several drug discovery lead compounds which may treat Leishmaniasis and Chagas disease. Similarly, this approach can be used for discovery of therapeutic compounds for treatment of other infectious diseases in the future.
- substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, at times 75% free, at times 90% free, and often 95% free from other components with which they are naturally associated or associated with by virtue of the purification process.
- Peptide and polypeptide are used interchangeably herein and refer to a compound made up of a chain of amino acid residues linked by peptide bonds. Unless otherwise indicated, the sequence for peptides is given in the order from the amino terminus to the carboxyl terminus.
- substitution refers to the replacement of one or more amino acids by different amino acids, respectively.
- Constant amino acid substitutions are substitutions which do not result in a significant change in the activity or tertiary structure of a selected polypeptide. Conservative amino acid substitutions may be made in the amino acid sequences to obtain derivatives of the peptides that may advantageously be utilized in the present invention.
- amino acids having aliphatic side chains including glycine, alanine, valine, leucine and isoleucine
- amino acids having non- aromatic, hydroxyl-containing side chains such as serine and threonine
- amino acids having acidic side chains such as aspartic acid and glutamic acid
- amino acids having amide side chains including glutamine and asparagine
- basic amino acids including lysine, arginine and histidine
- amino acids having aromatic ring side chains including phenylalanine, tyrosine and tryptophan
- amino acids having sulfur-containing side chains including cysteine and methionine.
- An "insertion” or “addition,” as used herein, refers to a change in an amino acid sequence with respect to a wild type amino acid sequence, resulting in the addition of one or more amino acid residues, as compared to the naturally occurring molecule.
- a “deletion,” as used herein, refers to a change in the amino acid sequence with respect to a wild type amino acid sequence, and results in the absence of one or more amino acid residues.
- a "variant" of a first amino acid sequence refers to a second amino acid sequence that has one or more amino acid substitutions or deletions, relative to the first amino acid sequence.
- a “modification" of an amino acid sequence or a “modified” amino acid sequence refers to an amino acid sequence that results from the addition of one or more amino acid residues, to either the N-terminus or the C-terminus of the sequence.
- a “modification” may also refer to a chemical modification to one or more amino acids within the peptide sequence, such as incorporation of an amino acid analog.
- the amino acid analog may be a naturally occurring analog or synthetic.
- modulate refers to a change in the binding between a LACK or TRACK protein and its cognate binding protein. Modulation or regulation may directly or indirectly cause an increase or a decrease in signal transduction activity, downstream binding characteristics, or any other biological, functional or immunological properties of the LACK or TRACK and/or its cognate binding protein.
- amino acid sequence having percent identity refers herein to an "amino acid sequence having percent identity" with another sequence intends that the sequences have the specified percent identity, , determined as set forth below, and share a common functional activity.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
- the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the length of the reference sequence.
- percent identity is taken as the number of like residues between the first and second sequence relative to the total number of residues in the longer of the first and second sequences.
- the comparison of sequences and determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
- the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol.
- Biol., 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blosum 80 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
- the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:1 1-17 (1989)) which has been incorporated into the LALIGN program (version 2.0), using a Blosum 80 weight residue table, a gap length penalty of 14 and a gap penalty of 4.
- Protein sequences can further be used as a "query sequence" to perform a search against public databases; for example, BLAST protein searches can be performed with the BLASTp program, max target sequences 100; expect threshold 200,000; matrix PAM30; gap costs existence: 9; extension: 1 . See worldwide web at: ncbi.nlm.nih.gov.
- Specific or “specificity” refers to the selective modulation of bioactivity by a modulatory peptide or peptide composition.
- a modulatory peptide can be tested for its specificity of modulation (inhibiting or activating) by comparing the amount of a downstream effect, such as, for example (but not limited to) inhibition of binding of LACK or TRACK to its cognate binding partner in the presence or absence of the modulatory peptide or peptide composition.
- the addition of a specific peptide inhibitor to a binding assay to measure binding of LACK or TRACK protein to its cognate binding protein in the presence and absence of the modulatory peptide results in a decrease in binding interaction between LACK or TRACK protein and its cognate binding protein.
- the decrease in binding interaction between the LACK or TRACK protein and its cognate binding proteinin the presence of modulator peptide is at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, or at least a 100-fold greater than the decrease in binding interaction between LACK or TRACK protein and its cognate binding protein when the modulator peptide is not present.
- LACK is highly homologous to the mammalian RACK1 (> 75% homology); however, there are regions of lower homology which may determine specific interactions of LACK with its binding partners. Five domains with low levels of amino acid conservation were identified. The non-conserved regions are mostly located in the outer most b-strand (b-strand-1 ) of each WD 40 domain.
- Peptides derived from the non-conserved regions were then synthesized and tested towards their biological activity in Leishmania parasites. Because peptides typically do not cross membranes, a TAT-derived peptide, TAT 47- 57 (Chen, 2001 ) has been used to introduce PKC regulating-peptides into cells. Conjugating a carrier peptide to a cargo peptide is a well-established delivery technology shown to be effective in mammalian cells in culture, in animals, as well as in human subjects (Gray, 1997; Inagaki, 2003; Inagaki, 2005; Chen, 2001 ; Inagaki, 2003; Roe, 2007).
- the TAT 47-57 delivery system has been successfully used to deliver an analogue of MARCKs-related protein effector domain (3DMRP E D), a substrate for gp63 (Leishmania protease) to both Leishmania promastigotes and macrophages (Corradin, 2002). Furthermore, a study with Plasmodium employed a 10 amino-acid peptide to show that a single dose of 250 ⁇ peptide inhibited 90% of parasite transformation (Dhawan, 2003 #1 1078). Thus, a similar system is used herein to deliver the peptides to the parasites and infected macrophages.
- LACK or TRACK protein and its cognate binding protein may be found in different sub-cellular locations.
- Downstream effectors in the PKC pathway can include, but are not necessarily limited to, myristoylated alanine-rich C-kinase substrate (MARCKS) (Disatnik et al., 2002, J. Cell Sci., 1 15:2151-2163; Myat et al., 1997, Curr. Biol. 7:61 1 -614), occludin (Qi et al., 2008, J. Clin. Inv., 1 18:173-182), and several ion channels (Barman et al., 2004, Am. J. Physiol. Lung Cell. Mol.
- c-Abl is on the endoplasmic reticulum (Qi et al., 2008, J. Cell Sci., 121 :804-813); dynamin-related protein 1 (Drp-1 ) on the mitochondria (Qi et al., 2010, Mol. Biol. Cell, 22:256-265); and pyruvate kinase and a heat shock protein (HSP27) are in the cytosol (Siwko et al., 2007, Int. J. Biochem. Cell Biol., 39:978-987).
- peptides that interfere with the auto-inhibitory interactions and thus act as activators of the corresponding isozyme have been identified (Chen et al., Proc. Natl. Acad. Sci. USA, 98:1 1 1 14-1 1 1 19; Ron et al., 1995, Proc. Natl. Acad. Sci. USA, 92:492-496).
- the inhibitors therein described were then used to show that 5PKC- mediated phosphorylation of PDK is required for 5PKC-dependent cardiac injury following an ischemic event.
- ⁇ inhibited 5PKC-mediated phosphorylation of PDK, but not the phosphorylation of other 5PKC substrates, such as MARCKS or Drp1. Its specificity for 5PKC was also evident by the absence of ⁇ effect in cells lacking 5PKC.
- ⁇ peptide represents a short sequence of similarity between PDK2, a direct substrate of 5PKC, and 5PKC. Like the ⁇ site, ALSTE, these peptides are all derived from the C2 domain. However, the action of ⁇ is different.
- the modulatory peptide comprises a carrier moiety and a linker moiety. In some embodiments, the modulatory peptide is directly linked to a carrier moiety without a linker moiety. In some embodiments, the modulatory peptide does not comprise a linker moiety or a carrier moiety. In some embodiments, the peptide moiety comprises a carboxamide or cyclic peptide.
- a composition comprising a modulatory peptide will reduce a binding interaction between a LACK or TRACK protein to its cognate binding partnerbut will not affect the binding interaction between the cognate binding partner and any other of its non-LACK or non-TRACK binding partners. It is understood that the effect of the modulatory peptide on the binding interaction between a LACK or TRACK protein to its cognate binding partner is merely to reduce the binding interaction between a LACK or TRACK protein and its cognate binding partner under equivalent assay conditions but in the absence of modulatory peptide.
- a “reduction” effect may encompass a decrease of 5%-20%, 10%-50%, 30%-50%, 40%-60%, 50%-80%, 70%- 90%, 80%-95%, or 90-99% in binding interaction between a LACK or TRACK protein and its cognate binding partner.
- this "reduction" effect encompasses at least a 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% decrease in binding interaction between a LACK or TRACK protein to its cognate binding partner.
- the selective modulatory peptides herein disclosed can inhibit protein-protein interactions between LACK or TRACK protein and its cognate binding protein.
- the peptides can comprise a core amino acid sequence which is similar to the LACK- or TRACK-binding protein, substrate or cargo peptide identified as described herein. This core amino acid sequence is at least 60% identical to the LACK- or TRACK-binding protein, substrate or cargo sequence.
- a peptide which can selectively inhibit interaction of the LACK or TRACK with its cognate binding partner may be significantly longer than this core sequence of 5 amino acid residues.
- a selective modulator peptide may be 5-20 amino acids in length, or 6-15 amino acids in length.
- the additional amino acids which may be N-terminal, C-terminal or both N-terminal and C-terminal to the core sequence are derived from a LACK- or TRACK-binding protein, substrate or cargo protein sequence. Accordingly, the entire length of the selective modulator peptide will be at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identical to a same length of sequence within the LACK- or TRACK-binding protein, substrate or cargo peptide for which the modulator peptide is effective.
- the peptides are described primarily with reference to amino acid sequences from Homo sapiens, it is understood that the peptides are not limited to the specific amino acid sequences set forth herein.
- the selective modulatory peptides can be used in native form or modified by conjugation to a carrier, such as those described below.
- a carrier such as those described below.
- one or two amino acids from the sequences can be substituted or deleted and exemplary modifications and derivatives and fragments for each peptide are given below.
- Deprotection was performed with a 20% piperidine in DMF with 0.1 M HOBt solution. Coupling reactions were performed with HBTU in DMF (0.1 1 M), amino acids in DMF (0.12 M) and DIEA in NMP solution (0.25 M). Each deprotection and coupling reaction was performed with microwave energy and nitrogen bubbling. Microwave cycle included two deprotection steps of 30 sec and 180 sec, each. All coupling reactions lasted 300 sec. Peptide cleavage from the resin and deprotection of the amino acids side chains were carried out with TFA/TIS/ H 2 0 /phenol solution (90:2.5:2.5:5 v/v/v/v/v) for 4 h at room temperature. The resin was washed with TFA.
- Products were purified by preparative reverse-phase high-pressure liquid chromatography (RP-HPLC; Shimadzu LC-20) equipped with CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 ⁇ LC-6AD solvent delivery unit and FRC-1 OA fraction collector from Shimadzu, MD, USA), using an XBridge Prep OBD C18 5pm (19 mm x 150 mm) column (Waters, MA, USA) at 10 mL/min.
- the solvent systems used were A (H 2 0 with 0.1 % TFA) and B (CH 3 CN with 0.1 % TFA). For separation, a linear gradient of 5-95% B in 45 min was applied and the detection was at 215 nm.
- Modulatory Peptide Compositions Comprising a Carrier Moiety
- the modulatory peptide useful in inhibiting LACK or TRACK protein-protein interactions with its cognate binding protein can be attached or linked to a peptide moiety which facilitates transfer of the modulatory peptide composition across a cell membrane.
- This peptide carrier may be any one of a number of peptide carriers known in the art for facilitating transfer across cell membranes, including Tat, the Drosophila Antennapedia protein, a polycationic peptide such as polyarginine or polylysine) (e.g., (R) 7 ), penetratin, Tat, VT5, MAP, Transportan, Transportan-10, pVEC, pISL, Pep-1 , and Mouse PrPC (1-28) (see Lundberg et al., 2003, J.
- the carrier peptide is Tat-derived transport polypeptide (U.S. Pat. Nos. 5,747,647 and 5,804,604; Vives, et al. J. Biol. Chem., 272:16010-16017 (1997)), polyarginine (U.S. Pat. Nos. 4,847,240 and 6,593,292; Mitchell et al., 2000; Rothbard et al., 2002) or Antennapedia peptide (U.S. Pat. No. 5,888,762). The disclosures of these references are incorporated herein in their entirety.
- the modulatory peptide may be linked to the carrier peptide by a disulfide bond.
- the disulfide bond is formed between two cysteines, two cysteine analogs or a cysteine and a cysteine analog.
- both the modulatory peptide and the carrier peptide contain at least one cysteine or cysteine analog.
- the cysteine residue or analog may be present as the N-terminal or C-terminal residue of the peptide or as an internal residue of the modulatory peptide and of the carrier peptide.
- the disulfide linkage is then formed between the sulfur residues on each of the cysteine residues or analogs.
- the disulfide linkage may form between, for example, the N-terminus of the modulatory peptide and the N-terminus of the carrier peptide, the C-terminus of the modulatory peptide and the C-terminus of the carrier peptide, the N-terminus of the modulatory peptide and the C-terminus of the carrier peptide, the C-terminus of the modulatory peptide and the N-terminus of the carrier peptide, or any other such combination including at any internal position within the modulatory peptide and/or the carrier peptide.
- the modulatory peptide can alternatively be part of a fusion protein.
- the peptide is bound to another peptide by a bond other than a Cys-Cys bond.
- An amide bond from the C-terminal of one peptide to the N-terminal of the other is exemplary of a bond in a fusion protein.
- This embodiment encompasses the presence of a peptide bond between and linking the modulatory and carrier peptides to form a single linear peptide composition comprising both the modulatory peptide and the carrier peptide.
- the modulatory peptide may be N-terminal to the carrier peptide, or the carrier peptide may be N-terminal to the modulatory peptide.
- a short linker peptide may be present between the modulatory peptide and the carrier peptide within the single linear peptide composition.
- the linker peptide may comprise 1 to 15 amino acids.
- the linker peptide may comprise 2 to 10 amino acids, 3 to 10 amino acids, 4 to 10 amino acids, 2 to 8 amino acids, 3 to 7 amino acids, or 4 to 6 amino acids.
- the linker peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids.
- the linker peptide comprises
- the linker peptide comprises 1 , 2, 3, 4, or 5 glycine residues.
- the linker peptide comprises 1 ,
- the linker peptide comprises at least 1 serine residue.
- the linker is Gly-Ser-Gly. It is understood that the linker peptide may comprise any amino acid or amino acid analog,
- the single linear peptide composition may alternative have a single amino acid present between the modulatory peptide and the carrier peptide.
- kinase modulators are very important for basic research as well as drugs. Numerous kinase modulators have been developed earlier. Most of these regulators are small molecules many with broad activity and other with higher selectivity (Karaman et al., 2008, Nat. Biotech., 26:127-132). That a modulator peptide can be specific for a single signaling molecule is surprising and novel. Such specific regulators can be rationally designed and these peptides provide missing tools to determine the role of one of several cellular functions of, for example, a given PKC isozyme. This approach is likely applicable to other signaling proteins, allowing the generation of separation-of function regulators of other protein-protein interactions.
- the modulatory peptides and peptide compositions described herein may be administered to a subject in need thereof to prevent or reduce infection by a parasite, e.g., Leishmania or Trypanosoma species. Such peptides are useful for slowing or inhibiting the progression of parasitic infection in a subject, such that the subject more closely resembles a healthy animal.
- a method of treating an individual at risk or with an established parasitic infection comprises the step of administering to the individual a pharmacologically effective amount of a modulatory peptide or composition that selectively inhibits LACK or TRACK protein-protein interactions with its cognate binding protein.
- an effective amount or “pharmacologically effective amount” refers to the amount of peptide or composition required to confer a therapeutic effect on the treated subject, e.g., reduced susceptibility to parasitic infection. Effective doses will also vary, as recognized by those skilled in the art, depending on the route of administration, the excipient usage, and the optional co-usage with other therapeutic treatments. In still yet another embodiment, there is a method of protecting the subject from parasitic infection.
- Such a method comprises administering a modulatory peptide composition wherein the administering results in increased protection of macrophages from parasitic infection, reduces apoptotic cell death caused by parasitic infection, as compared to said results in the absence of administering a modulatory peptide composition or administering a control peptide composition.
- the reducing can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease, or any value or range there between, in the amount of disease due to parasitic infection, including but not limited to lesion size.
- modulatory peptides and peptide compositions may be co-administered in a composition with a second therapeutic agent.
- the modulatory peptides individually, in combination, or combined with a second therapeutic agent, may be used to prepare a medicament for the slowing or inhibiting the progression of, for example, parasitic infection, leishmaniasis, trypanosomiasis, or Chagas disease.
- a pharmaceutical composition comprising a described compound and at least one pharmaceutically acceptable excipient or carrier.
- Methods of preparing such pharmaceutical compositions typically comprise the step of bringing into association a described compound with or without a carrier moiety and, optionally, one or more accessory ingredients.
- the described compounds and/or pharmaceutical compositions comprising same may be formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. Typically, formulations are prepared by uniformly and intimately bringing into association a described compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
- compositions of the present invention suitable for parenteral administration comprise one or more described compounds in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, amino acids, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
- aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
- polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
- vegetable oils such as olive oil
- injectable organic esters such as ethyl oleate.
- Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
- compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the described compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include agents to control tonicity, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
- adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
- a described compound may be delivered to a human in a form of solution that is made by reconstituting a solid form of the drug with liquid.
- This solution may be further diluted with infusion fluid such as water for injection, 0.9% sodium chloride injection, 5% dextrose injection and lactated ringer's injection.
- infusion fluid such as water for injection, 0.9% sodium chloride injection, 5% dextrose injection and lactated ringer's injection.
- the reconstituted and diluted solutions may be used within 4-6 hours for delivery of maximum potency.
- a described compound may be delivered to a human in a form of tablet or capsule.
- Injectable depot forms are made by forming microencapsulated matrices of the described compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
- the described compounds When the described compounds are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition may contain 0.2-25%, preferably 0.5-5% or 0.5-2%, of active ingredient.
- These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including, e.g., subcutaneous injection, subcutaneous depot, intravenous injection, intravenous or subcutaneous infusion. These compounds may be administered rapidly (within ⁇ 1 minute) as a bolus or more slowly over an extended period of time (over several minutes, hours or days). These compounds may be delivered daily or over multiple days, continuously or intermittently. In one embodiment, the compounds may be administered transdermally (e.g., using a patch, microneedles, micropores, ointment, microjet or nanojet).
- the described compounds which may be used in a suitable hydrated form, and/or the pharmaceutical compositions, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
- the selected dosage level will depend upon a variety of factors including the activity of the particular described compound employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
- a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
- the physician or veterinarian could start doses of the described compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
- a suitable daily dose of a described compound will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intramuscular, transdermal, intracerebroventricular and subcutaneous doses of the described compounds for a patient, when used for the indicated effects, will range from about 1 ⁇ g to about 5 mg per kilogram of body weight per hour. In other embodiments, the dose will range from about 5 ⁇ g to about 2.5 mg per kilogram of body weight per hour. In further embodiments, the dose will range from about 5 ⁇ g to about 1 mg per kilogram of body weight per hour.
- the effective daily dose of a described compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
- the described compound is administered as one dose per day.
- the compound is administered continuously, as through intravenous or other routes.
- the compound is administered less frequently than daily, such as every 2-3 days, in conjunction with dialysis treatment, weekly or less frequently.
- the subject receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
- the described compounds may be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides.
- Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
- route of administration is intended to include, but is not limited to subcutaneous injection, subcutaneous depot, intravenous injection, intravenous or subcutaneous infusion, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intraadiposal administration, intraarticular administration, intrathecal administration, epidural administration, inhalation, intranasal administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisternal administration and topical administration, transdermal administration, or administration via local delivery (for example by catheter or stent).
- Transdermal drug delivery to the body is a desirable and convenient method for systemic delivery of biologically active substances to a subject, and in particular for delivery of substances that have poor oral bioavailability, such as proteins and peptides.
- the transdermal route of delivery has been particularly successful with small (e.g., less than about 1 ,000 Daltons) lipophilic compounds, such as scopolamine and nicotine, that can penetrate the stratum corneum outer layer of the skin, which serves as an effective barrier to entry of substances into the body.
- small lipophilic compounds such as scopolamine and nicotine
- the viable epidermis which contains no blood vessels, but has some nerves. Deeper still is the dermis, which contains blood vessels, lymphatics and nerves. Drugs that cross the stratum corneum barrier can generally diffuse to the capillaries in the dermis for absorption and systemic distribution.
- the modulator peptide is delivered via microporation. Any one of a number of techniques for microporation is contemplated, and several are briefly described.
- Microporation can be achieved by mechanical means and/or external driving forces, to breach the stratum corneum to deliver the calcimimetic agents described herein through the surface of the skin and into the underlying skin layers and/or the bloodstream.
- the microporation technique is ablation of the stratum corneum in a specific region of the skin using a pulsed laser light of wavelength, pulse length, pulse energy, pulse number, and pulse repetition rate sufficient to ablate the stratum corneum without significantly damaging the underlying epidermis.
- the calcimimetic agent is then applied to the region of ablation.
- Another laser ablation microporation technique referred to as laser-induced stress waves (LISW)
- LISW involves broadband, unipolar and compressible waves generated by high-power pulsed lasers.
- the LISWs interact with tissues to disrupt the lipids in the stratum corneum, creating intercellular channels transiently within the stratum corneum. These channel, or micropores, in the stratum corneum permit entry of the calcimimetic agent.
- Sonophoresis or phonophoresis is another microporation technique that uses ultrasound energy.
- Ultrasound is a sound wave possessing frequencies above 20 KHz. Ultrasound can be applied either continuously or pulsed, and applied at various frequency and intensity ranges (Nanda et a/., Current Drug Delivery, 3:233 (2006)).
- microporation technique involves the use of a microneedle array.
- the array of microneedles when applied to a skin region on a subject pierce the stratum corneum and do not penetrate to a depth that significantly stimulates nerves or punctures capillaries. The patient, thus, feels no or minimal discomfort or pain upon application of the microneedle array for generation of micropores through which the modulatory agent is delivered.
- Microneedle arrays comprised of hollow or solid microneedles are contemplated, where the modulatory agent can be coated on the external surface of the needles or dispensed from the interior of hollow needles. Examples of microneedle arrays are described, for example, in Nanda et a/., Current Drug Delivery, 3:233 (2006) and Meidan et al. American J. Therapeutics, 1 1 :312 (2004). First generation microneedle arrays were comprised of solid, silicon microneedles that were externally coated with a therapeutic agent. When the microarray of needles was pressed against the skin and removed after about 10 seconds, the permeation of the agent on the needles into the body was readily achieved.
- Second generation microneedle arrays were comprised of microneedles of solid or hollow silicon, polycarbonate, titanium or other suitable polymer and coated or filled with a solution of the therapeutic compound. Newer generations of microneedle arrays are prepared from biodegradable polymers, where the tips of the needles coated with a therapeutic agent remain in the stratum corneum and slowly dissolve.
- the microneedles can be constructed from a variety of materials, including metals, ceramics, semiconductors, organics, polymers, and composites. Exemplary materials of construction include pharmaceutical grade stainless steel, gold, titanium, nickel, iron, tin, chromium, copper, palladium, platinum, alloys of these or other metals, silicon, silicon dioxide, and polymers.
- biodegradable polymers include polymers of hydroxy acids such as lactic acid and glycolic acid polylactide, polyglycolide, polylactide-co-glycolide, and copolymers with poly(ethylene glycol), polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide- co-caprolactone).
- Representative non-biodegradable polymers include polycarbonate, polyester, and polyacrylamides.
- the microneedles can have straight or tapered shafts.
- the diameter of the microneedle is greatest at the base end of the microneedle and tapers to a point at the end distal the base.
- the microneedle can also be fabricated to have a shaft that includes both a straight (untapered) portion and a tapered portion.
- the needles may also not have a tapered end at all, i.e. they may simply be cylinders with blunt or flat tips.
- a hollow microneedle that has a substantially uniform diameter, but which does not taper to a point, is referred to herein as a "microtube.”
- the term "microneedle" includes both microtubes and tapered needles unless otherwise indicated.
- Electroporation is another technique for creating micropores in the skin. This approach uses the application of microsecond or millisecond long high-voltage electrical pulses to created transient, permeable pores within the stratum corneum.
- microporation techniques include use of radio waves to create microchannels in the skin.
- Thermal ablation is yet another approach to achieve delivery of larger molecular weight compounds transdermally.
- Peptide synthesis Peptides were synthesized by conventional manual synthesis, or using Microwave by Liberty Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC, USA) or by American Peptide (CA, USA).
- peptides were conjugated to a TAT carrier (see below) by disulfide bond as described in Chen et al. (2001 , Chem. Biol., 8:1 123-1 129).
- no disulfide bridge is used, but rather, peptides are synthesized and connected to TAT as one polypeptide:
- the cargo is at the N terminus and TAT is at the C- terminus.
- the C-terminus of the peptides was modified to C(0)-NH 2 using Rink Amide AM resin to increase stability (as described in Sabatino et al. ⁇ Cur. Opin. in Drug Disc. & Dev., 1 1 :762-770).
- Peptides were analyzed by analytical reverse-phase high-pressure liquid chromatography (RP-HPLC) (Shimadzu, MD, USA) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and purified by preparative RP- HPLC.
- TAT also known as Tat.47.57 (RRRQRRKKRGY; hereinafter SEQ ID NO: 1 ) to enable their transmembranal delivery.
- RRRQRRKKRGY a peptide carrier
- the TAT peptide may also be used in the reverse orientation, (YGRKKRRQRRR; hereinafter SEQ ID NO: 2).
- Peptide 213 was synthesized as negative control and was not active.
- GG Glycine-Serine-Glycine tripeptide
- GSG Glycine-Serine-Glycine tripeptide
- b-Ala beta- Alanine
- GABA gamma-amino-n-butyric acid
- Fmoc-Epsilon-Ahx-OH Fmoc-Epsilon-Ahx-OH
- Leishmania viability in culture Cultures of different strains of parasites (causative of the 3 different clinical forms: L. major- cutaneous, L. donovani- visceral and L. braz/V/ens/s-muco-cutaneous), carried out in triplicates, were treated with different concentrations of peptides, using two protocols (1 ) a single dose at time 0 for one hour followed by 24 hrs of culturing or (2) parasites were treated with three treatments every 4 hours during the first hours of the study. The parasites were incubated with increasing concentrations (0-243 ⁇ ) of various peptides, and parasite proliferation and viability were evaluated by MTT assay after 2, 24, 48 and 72 hours (Sereno, 1997).
- Peptides had a marginal effect at very low concentration (3 ⁇ ), while they were almost 100% effective at moderate concentrations (81 ⁇ , peptide LL M -1 .4) and peptide (LL M -2.1 ) induced almost complete parasite cell death at a higher concentration (243 ⁇ ).
- the IC 50 of the most active peptides is considered low for a first screen for a lead compound.
- Peptides LL M -1.1 and LL M -2.2 were also tested in dose-response studies, and these peptides displayed higher trypanocidal activity. A strong correlation between concentration and trypanocidal activity was shown for these peptides.
- Trypanocidal activity of the peptides was tested against trypomastigotes forms. Trypomastigotes Y stain (2 x 10 6 ), obtained from 5° or 6° day of infection of cultures of LLC-MK 2 cell line (Andrews, 1982). The assay was carried out in fresh modified Eagle's medium (MEM) phenol red free with 2% FBS and incubated with different peptides at 100 ⁇ for two hours at 37 °C and 5% C0 2 . The disruption of parasite's motility was monitored by microscopic examination.
- MEM fresh modified Eagle's medium
- Parasite viability was measured by a colorimetric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium monosodium salt (WST-1 ) reagent (Roche). Cell viability was determined by the level of reduced WST-1 measured at 450 nm, expressed as a percentage of the control as a reference.
- Peptides IC 5 o assay The peptides with better trypanocidal activity (LL M -1.1 and LL
- Peptides derived from LACK or TRACK protein and homologous to regions in Trypanosoma cruzi (TcRACKI ) linked to Tat carrier peptide effectively killed trypomastigote forms of Trypanosoma cruzi in a dose responsive manner within one hour of treatment, independent of the linker. Peptide alone, without Tat, had no effect.
- Modulatory peptides 340e, 308, 329, 331 , 341 b and 341 d reduced parasite viability to 25-50%
- modulatory peptides 227, 343 and 309 reduced parasite viability to 50-75% of parasite viability absent treatment with modulatory peptide.
- the TAT peptide was used to deliver the peptides into the cell, and various linkers between the cargo and the TAT peptide were employed to test and optimize linker length. Addition of two Gly amino acids to the linker between peptide LL M -1.1 and the carrier peptide (GG, LL M -1.1 ) was found to be more effective against Leishmania amazonensis promastigotes compared to peptides with one amino acid spacer such as, beta-alanine (LLn-1.2), gamma-aminobutyric acid (Gaba) (LL
- TAT carrier peptide The location of the TAT carrier peptide was not important as was observed by comparing peptides LL M -1.4 in which the TAT sequence is at the N-terminus and LL M -1 .5 at the C-terminus. Moreover, peptide lacking TAT but containing the same cargo sequence (LL M -1.6) was found to have no cytotoxic effect at all, demonstrating the utility of TAT for cell delivery.
- peptides were assessed for efficacy in reducing the ability of promastigotes to infect macrophages.
- Promastigotes of L. amazonensis were incubated with peptides (0-200 ⁇ ) for 1 hour, parasites were then washed and used to infect macrophages. Infected cells were incubated for an additional 30 h without any further treatment.
- Peptide LL M -1.4 was the most effective peptide in this assay, resulting in over 75% reduction in the number of infected cells at 200 ⁇ .
- concentration for the peptides is somewhat high, promastigotes were treated only once prior to infection which was assessed after 30 h.
- peptides LL M -1.4 and LL M -2.1 had an effect not only on parasite viability but also on their ability to infect macrophages and to propagate in infected host cells, characteristics of great interest for development of therapeutic peptides.
- Peritoneal macrophages from BALB/c mice (susceptible to Leishmania infection) or a murine macrophage cell line, J774, adhered onto coverslips (Callahan, 1997) were infected with Leishmania parasites at a 1 :10 (macrophage to parasite ratio) at 34°C in a humidified environment containing 5% C0 2 .
- promastigotes were treated with the indicated peptides XX minutes prior to infection and cultures were maintained for an additional 96 h.
- Cells were fixed after the infection and stained with Giemsa for microscopical estimation of amastigote infection-rate/survival. The number of infected macrophages and the number of parasites per macrophage were determined.
- Cardiac myocytes were treated with 20 ⁇ peptides for 20 min followed by 10 nM PMA as indicated. Cells were lysed and the lysate was centrifuged at 800 g and then the nuclei discarded and the supernatant then spun at 100,000 g to obtain the particulate fraction in which the level of beta2 PKC was determined.
- the level of beta2 at the membrane is a marker of activation and translocation and dependent on the interaction of beta2 with RACK1.
- the level of beta2 PKC at the membrane in the presence of the peptides was determined. Beta V5-3 peptide, a known beta2 specific inhibitor, was used as a control. Note the increase of translocation of PKC in the presence of PMA with no peptide present of 2 fold.
- Neonatal cardiac myocytes were isolated from neonatal rats as previously described (Qi, 2007). Cardiac myocytes were isolated by using the cardiomyocyte isolation kit from Cellutron. Cardiac myocytes represent 90-95% of total adherent cells. Cells were maintained in MEM Eagle's with Earle's BSS media (containing 50 U/ml penicillin, 80 pmol/l vitamin B 12 , 0.1 mM bromodeoxyuridine (BrdU) and 80 pmol/l vitamin C) with 10% serum. After 4 days, the cells were treated with respective peptides at 1 mM for 15 min before adding 10 nM PMA for 30 min.
- MEM Eagle's with Earle's BSS media containing 50 U/ml penicillin, 80 pmol/l vitamin B 12 , 0.1 mM bromodeoxyuridine (BrdU) and 80 pmol/l vitamin C
- cardiac myocytes were washed with cold phosphate-buffered saline, scraped in homogenization buffer (Tris-HCI 50 mM, EGTA 1 mM, EDTA 1 mM and 250 mM sucrose), and lysed by passing the cell extract though a 26 G needle.
- the extract was spun at 100,000 x g for 30 min at 4 °C. The supernatants correspond to the cytosolic fraction.
- the pellets were resuspended in homogenization buffer containing 0.1 % Triton X-100 and correspond to the particulate fractions.
- ⁇ was immunoprecipitated from the particulate fractions as described previously (Disatnik, 2002) using anti-PKCpil antibody (Santa Cruz Biotechnology).
- the immunoprecipitated kinase of respective peptide treatment was incubated 30 min at 37 ° C in 40 ⁇ of binding buffer (20 mM Tris-HCI, 20 mM MgCI 2 , 1 mM DTT, 25 mM ATP) containing 5 pCi [ ⁇ 32 ⁇ ] ATP (4500 Ci/mmole, ICN) and 10 mM MBP. After incubation, assays were terminated by adding loading buffer containing 5% SDS. The samples were loaded on 14% SDS acrylamide gel and transferred to nitrocellulose. The level of phosphorylated MBP was analysed by exposing the nitrocellulose to autoradiography.
- the reaction was performed in presence of PKC activators, phosphatidylserine (60 pg/ml) and sn-1 ,2 dioleoylglycerol (2 pg/ml).
- 460 ul phophatidylserine and 16 ⁇ DG were added together and evaporated using nitrogen or argon.
- the mixture was dried and 4 ml Tris-Hcl 20 mM added, then the mixture was sonicated for three times at medium force. This preparation may be used for one month when stored at 4 ° C.
- Cell viability assay may be used for one month when stored at 4 ° C.
- LLC-MK2 cells were seeded in 96-well plates at a density of 8,000 cells/well 24 h prior to treatments. All cell treatments were carried out in MEM medium phenol red- free supplemented with 2% FBS. The cells received three doses of 100 ⁇ of each peptideo (LLII-1.1 , LLII-2.2) at time-point 0, 3 and 6 h, 12 h after the last addition of peptide, cell viability was determined using WST-1 (Roche, Indianapolis, IN) according to the manufacturer's protocol. Cell morphology was accessed by light microscopy.
- Macrophages were infected with promastigotes for 2 hours, cells were washed and then treated with peptides for 48 h, with changes in medium every 12 h.
- novel modulatory peptides disclosed herein were found to be effective against both the old and new world species. However, it is also understood that the treatment / modulatory peptide can be tailored to a particular Leishmania species.
- the rationally designed peptides described herein exhibited anti-parasitic effects, inducing parasite cell death, reducing parasite viability, protecting macrophages from parasite infection and reducing the number of macrophage infected by the parasite, while having no toxic effect on host or control cells. Furthermore, the requirement for the parasite to develop two or more mutations to avoid susceptibility to a drug agent also dramatically reduces chance of parasitic resistance. These LACK / TRACK inhibitor peptides were also found to be effective against T. Cruzi and Chagas disease.
- these peptide inhibitors are useful lead for the development of novel treatment of diseases, such as Cutaneous Leishmaniasis, Mucosal Leishmaniasis, Visceral
- Leishmaniasis and trypanosomiasis are diseases caused by related parasites that affect millions of people mainly in the poorest countries and rural areas. Very few new drugs are currently under development and in general the current treatment relies on old, often toxic and ineffective drugs.
- a modulatory peptide described herein bound elF1A previously described to be a TRACK-binding protein. This is of interest because TRACK has been shown to be involved in protein translation in Trypanosoma brucei and RNAi for TRACK in T brucei inhibited cytokinesis, perhaps by inhibiting the translation of a protein that aids cytokinesis.
- the peptides used in these studies were also found to be non-toxic, and were observed not to have an effect on non-infected macrophages or other cultured cells.
- inhibitory peptides disclosed herein have several advantages:
- the peptides can be used in a screen to identify small molecule inhibitors.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A peptide inhibitor is provided which specifically inhibits the binding of a LACK or TRACK protein to its cognate binding protein. The peptide inhibitor is useful in methods and compositions for protection against infection by Leishmania or Trypanosoma species, as well as for inhibition and treatment for neglected tropical diseases.
Description
COMPOSITIONS AND METHODS FOR SPECIFIC INHIBITION OF LEISHMANIA RECEPTOR FOR ACTIVATED C-KINASE (LACK)
STATEMENT REGARDING GOVERNMENT INTEREST
[0001] This invention was made with Government support under contracts AI078505, HL052141 , and TW008781 awarded by the National Institutes of Health. The Government has certain rights in this invention.
REFERENCE TO SEQUENCE LISTING
[0002] A Sequence Listing is being submitted electronically via EFS in the form of a text file, created August 27, 2013, and named 586008274seqlist.txt (32,768 bytes), the contents of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0003] This disclosure relates generally to rationally designed anti-parasitic peptide inhibitors of receptor for activated C-kinase (RACK) orthologs, known in Leishmania species as "LACK" and in Trypanosoma cruzi (T cruzi) as "TRACK," and methods of their use for protection against infection by parasites, as well as for inhibition and treatment for neglected tropical diseases, such as, for example Leishmaniasis and Chagas Disease.
BACKGROUND
[0004] Neglected tropical diseases (NTDs) pose challenges for drug discovery. Approximately 40% of the human population is affected by NTDs such as parasitic diseases; however, these diseases receive comparatively little attention, as they largely affect poor people in developing countries. In particular, leishmaniasis and trypanosomiasis affect millions of people mainly in poor countries and rural areas throughout the Americas. Trypanosoma cruzi is the causative agent of Chagas disease, a potentially life-threatening illness which is endemic in South and Central America, affecting approximately 12 million people in Latin America, resulting in an incidence of 21 ,000 deaths (Teixeira, 2006). The disease has been found in North America as well, and over 25 million worldwide are at risk of acquiring the disease (who.int/mediacentre/factsheets/fs340/en/index.html). Despite the positive impact of the Southern Cone Initiative on Triatoma infestans control, it is estimated that around 70 million people live in transmission risk areas (Moncayo, 2003). The drug used for the treatment of Chagas disease, benznidazole, has limited use due to high toxicity. In
addition, benznidazole is ineffective in the chronic phase, the period of disease when majority of diagnoses are made. Individuals with chronic Trypanosoma infection develop Chagas disease, and may develop other complications, such as cardiac alterations (30%) and digestive, neurological or mixed alterations (10%). Chagas disease is mostly transmitted to humans by the feces of triatominae bugs, sometimes known as 'kissing bugs,' depending on the geographical area. Furthermore, NTD complicity can be related to the sophisticated defense mechanisms that parasites have developed against mammalian immune system, demonstrated partially by lipophosphoglycans that are found on the parasite surface and used by the parasite to promote its survival in the host (Hutchinson, 2007; Barrett, 2007). At present, most treatments for neglected tropical diseases, are prohibitively expensive, typically require multiple doses by injection and commonly have unacceptable toxicity. Frequently, drug resistance also arises (Barrett, 2003; Bouteille, 2003; Bray, 2003). In the past 25 years, only approximately one percent of the drugs addressing NTDs reached the market (Trouiller, 2002). Therefore, it is of great value to identify and develop more effective, novel therapeutics that will be less toxic and relatively inexpensive (Liu, 1996; Frearson, 2007).
[0005] Trypanosomatids are protozoan parasites distinguished by having only a single flagellum. The name is derived from the Greek trypano (borer) and soma (body), indicative of the corkscrew-like motion of some trypanosomatid species. Trypanosomatid parasites are found primarily in insects; however, a few genera have life-cycles involving a secondary host, which may be a vertebrate, invertebrate or plant. These include several species that cause three major diseases in humans: African trypanosomiasis ("sleeping sickness" caused by Trypanosoma brucei), South American trypanosomiasis ("Chagas Disease" caused by Trypanosoma cruzi), and leishmaniasis (a set of trypanosomal diseases caused by various species of Leishmania).
[0006] Leishmania is a genus of Trypanosomatid protozoa, and is the parasite responsible for the disease leishmaniasis. The disease is transmitted by the bite of female sandflies of the genera Phlebotomus in the Old World and Lutzomyia in the New World. For example, New World Lutzomyia sandflies transmit the Leishmania amazonensis parasite, and Old World Phlebotomus transmit Leishmania donovani, whose primary hosts are vertebrates. Leishmania commonly infects hyraxes, canids, rodents, and humans. Leishmaniasis is the second-largest parasitic killer in the world, and currently affects 12 million people in tropical and subtropical regions of over 80 countries, with 1.5 to 2 million new cases occurring annually, and 59,000 deaths occurring in 2001 alone (See who.int/tdr/diseases/leish/diseaseinfo.htm). To date, no vaccines are commercially available for leishmaniasis and the current treatment is toxic;
furthermore, drug resistance to the most commonly used medication has been reported (Singh, 2003).
[0007] Described herein is an alternative approach to rational drug design, which focuses on modifying protein-protein interactions in parasites. Effective signal transduction pathways rely on correct distribution of regulatory proteins within the cell. Anchor proteins have been described to serve as scaffolds upon which signal complexes can assemble, helping to provide spatial organization to signal transduction processes by allowing the formation of multimeric complexes at appropriate locations in the cell. Association with different anchors allows some signal kinases to participate in and distinguish between multiple pathways. For example, Protein kinase C ("PKC") interacts with lipid-derived second messengers (and in some cases, calcium) which induces conformational changes to transform the enzyme from an inactive to an active state. Various activated PKC isoforms interact with various Receptors for Activated C-Kinase ("RACKs").
[0008] The Receptor for Activated C-Kinase 1 (RACK1 ) is a ubiquitous and highly conserved scaffold protein having WD repeats, and it acts an anchoring protein of ΡΚΟβΙΙ, helping to regulate a range of cell activities including cell growth, survival, differentiation, shape and protein translation by recruiting signal proteins to specific nuclear or plasma membrane sites, to the cytoskeleton or to the 40S ribosome. RACK1 forms productive ternary complexes with a wide range of signal protein partners; target proteins interact with RACK1 through SH2 domains, PH domains, C2 domains, and other specific sequences. Different isoforms of activated PKCs interact with various RACKs, which thereby mediate differential subcellular targeting of the PKC isoforms (Ron et a/., 1999, J. Biol. Chem., 274:27039-27046; Mochly-Rosen, 1995, Science, 268:247-251 ). The C2 domain in the regulatory region of PKC was found to mediate at least some of their binding to RACKs (Smith et a/., 1992, Biochem. Biophys. Res. Commun., 188:1235-1240; Johnson et a/., 1996, J. Biol. Chem., 271 :24962-24966). Unique sequences within the highly conserved C2 domain (e.g., 3C2-4, 5V1-1 and eV1- 2) in each PKC isozyme are part of these interaction sites (Ron et a/., 1995, J. Biol. Chem., 270:24180-24187; Chen et a/., Proc. Natl. Acad. Sci. USA, 98:1 11 14-1 1 1 19; Gray et a/., 1997, J. Biol. Chem., 272:30945-30951 ). Peptides representing these unique sequences (e.g., 5V1-1 ) have been found to act as competitive inhibitors, disrupting the protein-protein interaction between the PKC isozyme and its corresponding RACK, thereby inhibiting the functions of a given PKC isozyme.
[0009] Alternatively, inhibitory intra-molecular protein-protein interactions have been observed to keep the enzyme in the inactive state. At least one such intra-molecular
interaction has been identified between the RACK-binding site in PKC and a sequence in the inactive form of a PKC isozyme which has amino acid sequence similarity to a site in the corresponding RACK; thus, the inactive PKC occludes the RACK binding site with a peptide sequence known as the pseudo-RACK ^RACK) (Dorn et al., 1999, Proc. Natl., Acad. Sci., 96:12798-12803). A peptide corresponding to this l+JRACK site competes with the intra-molecular inhibitory interaction, thus serving as a selective activator of the corresponding isozyme.
[0010] RACK1 is ubiquitous and highly conserved scaffold protein found in various species, including plants, parasites and yeast (Enserink, 2000; McCahill, 2002). RACK1 has been shown to be involved in many signaling processes leading to cell survival, growth and differentiation (reviewed in Schechtman, 2001 ). In Schizosaccharomyces pombe, the RACK1 homolog (Cpc2) associates with the ribosome, is essential for yeast metabolism. (Hoffmann, 1999) growth regulation, differentiation, and cell cycle progression. Yeast that do not express Cpc2 are viable but have a longer cell cycle (Shor, 2003; McLeod, 2000). Cpc2, like RACK1 , is a scaffold protein that binds and regulates the activity of key signaling molecules such as a PKC yeast homolog Pck2 (Won, 2001 ) and Rani (Pat1 ) kinase (involved in cell cycle progression) (McLeod, 2000).
[0011] A RACK1 ortholog has been identified in Leishmania, and has been dubbed "LACK" (Leishmania homologue of receptors for activated C kinase, RACK; also known as Leishmania activated C kinase receptor homologue) (Mougneau, 1995). LACK is found in both the amastigotes and promastigotes, and because of its homology to RACK, LACK is also believed to be a scaffolding protein, interacting with multiple signaling enzymes in these parasites, involved in essential signaling processes. In Leishmania major, there are four LACK genes encoding identical proteins, which appear to play an important role in the life of the parasite, as genetic knockouts of LACK are not viable (Locksley et al.), and parasites containing a single copy of the gene and expressing low levels of LACK fail to parasitize even immunocompromised mice (Kelly, 2003). LACK is found in a multiprotein complex in the parasite kinetoplast, likely bound to signaling enzymes involved in DNA replication (Gonzalez-Aseguinolaza, 1999). In Trypanosoma brucei (T. brucei), the RACK1 homologue ("TbRACKI ;" also known as Trypanosome activated C kinase receptor homologue or "TRACK") TbRACKI is a component of the translation machinery. When RNA interference was used to inhibit TbRACKI , initiation of translation and phosphorylation of a ribosomal protein were inhibited (Regmi, 2008). Furthermore, TRACK is required for cytokinesis, and
knockdown of TRACK impaired parasite growth (Rothberg, 2006). Infected mice with depleted TbRACKI caused elimination of circulating blood forms.
[0012] In the case of trypanosomes, cryo-electron microscopy failed to identify RACK1 on the 40S ribosome, suggesting that the trypanosome RACK1 (TRACK) may function in pathways other than translation.
[0013] Very few new drugs have been developed for treatment and/or prevention of these parasitic diseases. Current chemotherapies for leishmaniasis and trypanosomiasis are quite toxic, and drug resistance to the most commonly used medication has been reported. To develop novel therapeutics for treatment of leishmaniasis and trypanosomiasis, researchers are attempting to tailor drugs to target particular protozoan species, and/or to disrupt the interaction between certain proteins involved in their disease processes. The presently described approach to discovery of novel antiparasitic drugs focuses on modulating protein-protein interactions in parasites. This approach minimizes the possibility of development of resistance to inhibitors of protein- protein interactions.
[0014] Because protein-protein interactions are central to many biological processes, often determining the specificity of cellular signaling events, agents that modulate protein-protein interaction represent an important class of drugs. However, small molecules that interfere with protein-protein interactions are typically too small to cover the interface area between proteins (usually large, flat and noncontiguous); furthermore, small molecules often lack specificity and commonly cause side effects. Finding small- molecule inhibitors of protein-protein interaction has proven to be a challenge, as protein-protein interaction sites are often large and flat interfaces (750-1500 A) in each protein, rather than small, relatively "drugable" hydrophobic pockets (Arkin et a/., 2004, Nat. Rev. Drug Discov., 3:301-317). Thus, a long-felt need remains for inhibitors of parasite infections, as well as for novel tools to study the infectivity and viability of parasites.
[0015] Other compounds, such as proteins and peptides, are being pursued to interfere with protein-protein interactions, and rational drug design has been successfully used to develop short peptide inhibitors of protein-protein interactions between signaling enzymes and their scaffold proteins. Compared with proteins, therapeutic peptides have the potential to cross biological membranes due to their smaller size and are generally less immunogenic (McGregor, 2008). Additional advantages of peptides as drug candidates include lower manufacturing costs, higher activity per unit mass, greater specificity of action, and longer shelf life (Ladner, 2004; Vlieghe, 2010; Qvit, 201 1 ). These short bioactive peptides have been shown to be highly selective and effective in
several animal models of human diseases, and some of these peptides have been tested in humans and shown to be clinically safe.
[0016] LACK and TRACK have been shown to be essential for parasite survival/infectivity; thus, in the course of the present disclosure, LACK and TRACK were selected as drug targets, and certain regions of these parasite scaffold proteins involved in protein-protein interactions were identified and peptides derived from these regions were designed. Provided and disclosed herein are rationally designed peptides which act as selective inhibitors of LACK or TRACK activity and inhibit LACK / TRACK interactions with their cognate binding protein (e.g., a LACK-binding protein (LACK-BP). These selective peptide inhibitors of LACK / TRACK were conjugated to TAT-derived peptide for drug delivery, and a number of them were found to exert profound effects on parasites (inhibiting L. amazonensis promastigotes parasite viability and inducing parasite cell death (IC50 ~ 10 μΜ)), as well as the parasite's ability to infect macrophages, while the peptides themselves were found to be non-toxic to macrophages.
[0017] These peptide inhibitors provide the basis for prophylactic and/or therapeutic compositions for protection against infection by Leishmania species, as well as providing useful leads in the development of novel inhibitors for treatment for neglected tropical diseases (NTD) such as Cutaneous Leishmaniasis, Mucosal Leishmaniasis, Visceral Leishmaniasis and Chagas disease. The novel peptides of the present disclosure are effective against a variety of Leishmania species from both Old and New worlds. Furthermore, the methods described herein are very likely applicable in the rational design of other anti-parasitic drugs.
[0018] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.
BRIEF SUMMARY
[0019] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.
[0020] In one aspect, a peptide that selectively modulates the binding of a LACK or TRACK protein to its cognate binding proteinis provided. In some aspects, the modulatory peptide comprises a contiguous sequence of 5 amino acid residues, wherein said contiguous sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ I D NO: 10, SEQ ID NO:1 1 , SEQ I D N0: 12, SEQ ID N0:13, SEQ ID N0:14 SEQ ID NO: 15, SEQ I D NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ I D NO: 19, SEQ I D NO: 20, SEQ ID NO: 21 , SEQ I D NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ I D NO: 25, SEQ I D NO: 26, SEQ ID NO: 27, SEQ I D NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ I D NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. In some embodiments, the modulatory peptide consists of 5-20 amino acid residues. In some embodiments, the modulatory peptide is selected from the group consisting of SEQ I D NO: 3, SEQ I D NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ I D NO: 8, SEQ I D NO: 9, SEQ ID NO: 10, SEQ I D NO: 1 1 , SEQ I D NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ I D NO: 21 , SEQ I D NO: 22, SEQ I D NO: 23, SEQ I D NO: 24, SEQ I D NO: 25, SEQ ID NO: 26, SEQ I D NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ I D NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ I D NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. In some embodiments, provided herein is a selective modulatory composition comprising the peptide. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.
[0021 ] In some embodiments, a modulatory peptide (also known as a "cargo" peptide) is provided, wherein the peptide is linked to a carrier peptide sequence to enable transmembranal delivery. In some embodiments, the carrier peptide is a TAT peptide based on the Tat protein of Human Immunodeficiency Virus (HIV). In some embodiments, the carrier peptide is TAT47-57. In some embodiments, the carrier peptide is at least 60% identical to the sequence RRRQRRKKRGY (herein set forth as SEQ ID NO:1 ). In some embodiments, the carrier peptide is the sequence RRRQRRKKRGY (SEQ I D NO:1 ). In some embodiments, the amino acid sequence of the TAT peptide is reversed with respect to the amino and carboxy termini of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the carrier peptide is at least 60% identical to the sequence YGRKKRRQRRR based on the Tat protein of Human Immunodeficiency Virus (HIV) (SEQ ID NO:2). In some embodiments, the carrier peptide is the sequence YGRKKRRQRRR (SEQ ID NO:2).
[0022] In some embodiments, the modulatory peptide is linked to a spacer. In some embodiments, the spacer is a glycine-glycine dipeptide ("GG"). In some embodiments, the spacer is a glycine-serine-glycine tripeptide ("GSG"). Other linkers (also known as "spacers") are a beta-Alanine ("β-Ala" or "b-Ala"), cysteine (Cys), gamma-amino-n-butyric acid (also known as "GABA" or "Gaba"), or Fmoc-Epsilon-Ahx-OH. In some embodiments, the modulatory peptide is not linked to a spacer. In some embodiments, the modulatory peptide is linked to a cargo peptide, directly or indirectly. In some embodiments, the modulatory peptide is linked to a spacer which is linked to a cargo peptide. In some
embodiments, the carrier sequences (e.g., SEQ ID NO: 1 or SEQ ID NO: 2) are used as the cargo component. In some embodiments, a cargo sequence (e.g., SEQ ID NO: 3) is used as a carrier component. In some embodiments, the carrier peptide further comprises a sulfur-containing residue, which can be useful for conjugation. In some embodiments, the sulfur-containing residue is cysteine. In some embodiments, the sulfur-containing residue is a cysteine analog. In some embodiments, the carrier peptide further comprises a cysteine residue attached via a peptide bond to its C-terminus or N- terminus. In some embodiments, a cysteine residue is present as an alternative residue at any position within the carrier peptide or the cargo peptide.
[0023] In one embodiment, the modulatory peptide comprises of 5-20 amino acid residues. In another embodiment, the modulatory peptide comprises of 5-15 amino acids, 5-10 amino acids, 6-15 amino acids, 6-10 amino acids, or 6-8 amino acids. In still another embodiment, the modulatory peptide comprises 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 1 1 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, or 15 amino acids.
[0024] In some embodiments, the modulatory peptide is selected from the group consisting of CVSLAHATD (SEQ ID NO: 3), RNGQCQRK (SEQ ID NO: 5), MHEFLRD (SEQ ID NO: 6), NVIRVWN (SEQ ID NO: 7), VNGGKCERTLK (SEQ ID NO: 8), STGEQLFKINVESP (SEQ ID NO: 9), TPDGAKPSE (SEQ ID NO:10), RSLSVYD (SEQ ID NO: 1 1 ), QKKGDITDPYVKL (SEQ ID NO: 12), KTSVVRNN (SEQ ID NO: 13), GLNPYWMET (SEQ ID NO: 14), (QQAGSYIKVV) SEQ ID NO: 21 , (RHSVDSDYGLPSH) SEQ ID NO: 22, (EGHLKGHRGW) SEQ ID NO: 23, (DGTAIS) SEQ ID NO: 24, (ANPDRHSVS) SEQ ID NO: 25, (GLPSHRLE) SEQ ID NO: 26, (TGFVSC) SEQ ID NO: 27, (RSIRMWD)SEQ ID NO: 28, (CQRKFLK) SEQ ID NO: 29, (WVSSICFSPSLEH) SEQ ID NO: 30, (NVESPINQIA) SEQ ID NO: 31 , (KPSECISIAW) SEQ ID NO: 32, (GHKDNL) SEQ ID NO: 33, (DVVINSDGQ) SEQ ID NO: 34, and (NAGVTVRS) SEQ ID NO: 35.
[0025] In some embodiments, the modulatory peptide is not (SEQ ID NO: 3), (SEQ ID NO: 5), (SEQ ID NO: 6), (SEQ ID NO: 7), (SEQ ID NO: 8), (SEQ ID NO: 9), (SEQ ID NO:10), (SEQ ID NO: 1 1 ), (SEQ ID NO: 12), (SEQ ID NO: 13), (SEQ ID NO: 14), SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35.
[0026] In some embodiments, the modulatory peptide is at least about 60% identical to a contiguous sequence of equal length of a peptide sequence occurring in or derived from a substrate or cargo protein such as, for example (but not limited to) calmodulin A
from Trypanosoma brucei (T. brucei) (herein set forth as SEQ ID NO: 15), calmodulin (e.g., SEQ ID NO: 16), heat shock protein from T. cruzi (herein set forth as SEQ ID NO: 17), elongation factor from T. cruzi (herein set forth as SEQ ID NO: 18) or heat shock protein 70 from T. cruzi (herein set forth as SEQ ID NO: 19). In another embodiment, the modulatory peptide is at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a contiguous sequence of equal length derived from said protein or peptide.
[0027] In some embodiments, the modulatory peptide acts indirectly to inhibit activity of a downstream effector of a Protein Kinase C (PKC) isoform. In one embodiment, the modulatory peptide further comprises a sulfur-containing residue. In another embodiment, the sulfur-containing residue is cysteine. In still another embodiment, the sulfur-containing residue is a cysteine analog.
[0028] In one embodiment, the sulfur-containing residue is located at the N-terminus and/or the C-terminus of the modulatory peptide. In another embodiment, the sulfur- containing residue is an internal residue.
[0029] In one embodiment, the modulatory peptide is linked to a carrier peptide. In one embodiment, the modulatory peptide is linked to a carrier peptide by a disulfide bond. In another embodiment, the modulatory peptide is linked to the carrier peptide by a peptide bond, wherein the modulatory peptide and the carrier form a single modulatory fusion peptide.
[0030] In some aspects, a method for treating a subject comprising administration of a peptide or composition that selectively modulates the binding of a LACK or TRACK to its cognate binding partner is provided. In some embodiments, the subject is suffering from cutaneous leishmaniasis, mucosal leishmaniasis, visceral leishmaniasis, Chagas disease and/or trypanosomiasis. In some embodiments, the method comprises administering the modulatory peptide to a subject in need thereof. In other embodiments, the subject is undergoing or has undergone treatment with another antiparasitic agent.
[0031] In some aspects, provided herein is a method for reducing parasite infectivity comprising administering to a subject in need thereof, a peptide that selectively modulates the binding of a LACK or TRACK to its cognate binding partner. In some embodiments, a method for modulating the activity of effectors downstream of a PKC is provided.
[0032] These and other objects, features and additional embodiments of the present methods and compositions will become more fully apparent when read in conjunction with the following detailed description, drawings, examples, and claims. As can be
appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present invention. Additional aspects and advantages of the present invention are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figures 1A and 1 B illustrate exemplary modulatory peptides comprising SEQ ID NO: 3.
[0034] Figure 2 shows exemplary modulatory peptides comprising SEQ ID NO: 5.
[0035] Figure 3 shows exemplary modulatory peptides comprising SEQ ID NO: 10.
[0036] Figure 4 illustrates an exemplary in vivo peptide study protocol.
[0037] Figures 5-9 present the results of in vivo experiments with several exemplary modulatory peptides.
[0038] Figure 10 summarizes the effects of a modulatory peptide on parasite viability in a mouse model system.
BRIEF DESCRIPTION OF THE SEQUENCES
[0039] In one embodiment, the carrier peptide comprises an amino acid sequence identified by SEQ ID NO: 1.
[0040] In one embodiment, the carrier peptide comprises an amino acid sequence identified by SEQ ID NO: 2.
[0041] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 3.
[0042] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 4.
[0043] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 5.
[0044] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 6.
[0045] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 7.
[0046] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 8.
[0047] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 9.
[0048] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 10.
[0049] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 1 1.
[0050] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 12.
[0051] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 13.
[0052] In one embodiment, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 14.
[0053] In some embodiments, the LACK- or TRACK-binding protein is SEQ ID NO: 15.
[0054] In some embodiments, the LACK- or TRACK-binding protein is SEQ ID NO: 16.
[0055] In some embodiments, the LACK- or TRACK-binding protein is SEQ ID NO: 17.
[0056] In some embodiments, the LACK- or TRACK-binding protein is SEQ ID NO: 18.
[0057] In some embodiments, the LACK- or TRACK-binding protein is SEQ ID NO: 19.
[0058] In some embodiments, the LACK- or TRACK-binding protein is SEQ ID NO: 20.
[0059] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 21.
[0060] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 22.
[0061] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 23.
[0062] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 24.
[0063] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 25.
[0064] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 26.
[0065] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 27.
[0066] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 28.
[0067] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 29.
[0068] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 30.
[0069] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 31.
[0070] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 32.
[0071] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 33.
[0072] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 34.
[0073] In some embodiments, the cargo peptide comprises an amino acid sequence identified by SEQ ID NO: 35.
DETAILED DESCRIPTION
[0074] The Leishmania parasite life cycle includes two forms: non-motile amastigotes and flagellate promastigote forms. Shortly after a vector (e.g. sandfly) becomes infected with amastigotes during a blood meal from an infected reservoir host (e.g. human or other mammalian), the amastigotes are released into the vector's stomach. The parasites transform into promastigote forms inside the vector, and multiply in the extracellular blood and tissue fluids. The infected vector next feeds on a host by piercing the skin using his proboscis and injecting saliva which also contains anti-coagulant to avoid blood clotting, and the parasite. As soon as promastigotes infect the host, they are taken up by macrophages and the parasites revert from promastigotes to amastigote forms and the cycle continues.
[0075] Very few studies have been aimed at targeting parasite signaling processes; most drugs developed for infectious diseases are aimed at targeting host-pathogen interactions (Lira, 2001 ) or parasite metabolic pathways. Some studies have been aimed at developing new therapeutics for infectious diseases by targeting the catalytic site of
parasite kinases and metabolic enzymes; however, most of these have been unsuccessful, possibly due to the fact that targeting an enzyme's catalytic activity may favor development of drug resistance via mutations altering drug binding-sites without affecting biological function of the drug-target (Hastings, 2001 ). Targeting the catalytic site of an enzyme is also problematic because most catalytic sites of kinases are highly conserved and thus, inhibitors that lack specificity may affect host enzymes and signaling pathways (Hanks, 1988; Donald, 2002).
[0076] The present disclosure provides a different approach aimed at interfering with protein-protein interactions between the scaffold proteins homologous to RACK1 and their cognate binding partners, i.e., the Leishmania homologue Leishmania receptor for activated C kinase (LACK) and the Trypanosoma homologue, Trypanosoma receptor for activated C kinase (TRACK) with their binding partners. LACK is usually found in a multi- protein complex, was shown to play an important role in the early phase of Leishmania infection, and is required for parasite viability and for infection of the mammalian host (Mougneau, 1995). LACK-deficient parasites are not viable (Kelly, 2003), and parasites expressing lower levels of LACK fail to parasitize even immune compromised-mice. LACK also scaffolds a multi-protein complex in the parasite (Gonzalez-Aseguinolaza, 1999) and it is involved in parasite signaling processes and protein synthesis (Choudhury, 201 1 ).
[0077] In Trypanosoma brucei (Tbrucei) TRACK is expressed throughout the parasite life cycle localized predominantly in the perinuclear region and cytoplasm and has a role in the final stages of mitosis. Parasites with TRACK expression reduced by RNAi remained metabolically active, but were growth-impaired. Growth arrest in bloodstream forms was due to a delay in the onset of cytokinesis, and to an incomplete cytokinesis in procyclic forms. These parasites were eliminated from peripheral blood within 3 days post-infection. TRACK has also been shown to bind to eEF1A and is a component of the Tbrucei translational apparatus.
[0078] Both TRACK and LACK are conserved amongst different Trypanosoma and Leishmania species, and there is high homology between parasitic species, while there is less homology between TRACK or LACK and mammalian RACK1 , suggesting that certain peptide inhibitors of protein-protein interactions could be effective against both Trypanosome and Leishmania species while not affecting mammalian host proteins. The rationally designed peptides developed herein were based on the homology and structure of LACK and TRACK to RACK1 , and were found to bind a previously described TRACK binding protein, elF1A, as well as to dramatically decrease the viability and infectivity of parasites. A structure activity relationship study based on these lead
compounds allowed the identification of those exhibiting an increase in peptide activity against the parasite, reducing the number of viable free forms and intracellular parasites. As described herein, the approach previously used to inhibit PKC-scaffold protein interaction and consequently PKC signaling was also effective in inhibiting the interaction of this parasite scaffold protein with its binding partners, resulting in parasite death. Thus, the modulatory peptides described herein represent several drug discovery lead compounds which may treat Leishmaniasis and Chagas disease. Similarly, this approach can be used for discovery of therapeutic compounds for treatment of other infectious diseases in the future.
[0079] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.
[0080] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g.; A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Morrison and Boyd, Organic Chemistry (Allyn and Bacon, Inc., current addition); J. March, Advanced Organic Chemistry (McGraw Hill, current addition); Remington: The Science and Practice of Pharmacy, A. Gennaro, Ed., 20th Ed.; Goodman & Gilman The Pharmacological Basis of Therapeutics, J. Griffith Hardman, L. L. Limbird, A. Gilman, 10th Ed. "Synthesis of peptides and peptidomimetics" Methods of organic chemistry (Houben-Weyl) : additional and supplementary volumes to the 4th edition, 2004, Goodman, Murray; Toniolo, Claudio; Moroder, Luis; Felix, Aurthur; Thieme Medical Publishers Inc.
[0081] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 % to 8% is stated, it is intended that 2%, 3%, 4%, 5%, 6%, and 7% are also explicitly disclosed, as well as the range of values greater than or equal to 1 % and the range of values less than or equal to 8%. Similarly, if a range of 1 μηη to 8 μηη is stated, it is intended that 2 μηη, 3 μηη, 4 μηη, 5 μηη, 6 μηη, and 7 μηη are also explicitly disclosed, as well as the range of values greater than or equal to 1 μηη and the range of values less than or equal to 8 μηη.
I. Definitions
[0082] As used in this specification and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide" includes a single peptide as well as two or more of the same or different peptides, reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, and the like.
[0083] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the methodologies which are reported in the publications which might be used in connection with the invention.
[0084] The term "substantially purified", as used herein, refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, at times 75% free, at times 90% free, and often 95% free from other components with which they are naturally associated or associated with by virtue of the purification process.
[0085] "Peptide" and "polypeptide" are used interchangeably herein and refer to a compound made up of a chain of amino acid residues linked by peptide bonds. Unless otherwise indicated, the sequence for peptides is given in the order from the amino terminus to the carboxyl terminus. A "substitution", as used herein, refers to the replacement of one or more amino acids by different amino acids, respectively. "Conservative amino acid substitutions" are substitutions which do not result in a significant change in the activity or tertiary structure of a selected polypeptide. Conservative amino acid substitutions may be made in the amino acid sequences to obtain derivatives of the peptides that may advantageously be utilized in the present invention. Conservative amino acid substitutions, as known in the art and as referred to herein, involve substituting amino acids in a protein with amino acids having similar side chains in terms of, for example, structure, size and/or chemical properties. For example, the amino acids within each of the following groups may be interchanged with other amino acids in the same group as follows: amino acids having aliphatic side chains, including glycine, alanine, valine, leucine and isoleucine; amino acids having non- aromatic, hydroxyl-containing side chains, such as serine and threonine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; amino acids having amide side chains, including glutamine and asparagine; basic amino acids, including
lysine, arginine and histidine; amino acids having aromatic ring side chains, including phenylalanine, tyrosine and tryptophan; and amino acids having sulfur-containing side chains, including cysteine and methionine. Additionally, aspartic acid, glutamic acid and their amides, are also considered interchangeable herein.
[0086] An "insertion" or "addition," as used herein, refers to a change in an amino acid sequence with respect to a wild type amino acid sequence, resulting in the addition of one or more amino acid residues, as compared to the naturally occurring molecule.
[0087] A "deletion," as used herein, refers to a change in the amino acid sequence with respect to a wild type amino acid sequence, and results in the absence of one or more amino acid residues.
[0088] A "variant" of a first amino acid sequence refers to a second amino acid sequence that has one or more amino acid substitutions or deletions, relative to the first amino acid sequence.
[0089] A "modification" of an amino acid sequence or a "modified" amino acid sequence refers to an amino acid sequence that results from the addition of one or more amino acid residues, to either the N-terminus or the C-terminus of the sequence. A "modification" may also refer to a chemical modification to one or more amino acids within the peptide sequence, such as incorporation of an amino acid analog. The amino acid analog may be a naturally occurring analog or synthetic.
[0090] The term "modulate" or "regulate," as used herein, refers to a change in the binding between a LACK or TRACK protein and its cognate binding protein. Modulation or regulation may directly or indirectly cause an increase or a decrease in signal transduction activity, downstream binding characteristics, or any other biological, functional or immunological properties of the LACK or TRACK and/or its cognate binding protein.
[0091] Reference herein to an "amino acid sequence having percent identity" with another sequence intends that the sequences have the specified percent identity, , determined as set forth below, and share a common functional activity. To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 85%, 90%, or 95% of the length of the reference sequence. For the relatively short peptide sequences described herein, percent identity
is taken as the number of like residues between the first and second sequence relative to the total number of residues in the longer of the first and second sequences. The comparison of sequences and determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol., 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blosum 80 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:1 1-17 (1989)) which has been incorporated into the LALIGN program (version 2.0), using a Blosum 80 weight residue table, a gap length penalty of 14 and a gap penalty of 4. Protein sequences can further be used as a "query sequence" to perform a search against public databases; for example, BLAST protein searches can be performed with the BLASTp program, max target sequences 100; expect threshold 200,000; matrix PAM30; gap costs existence: 9; extension: 1 . See worldwide web at: ncbi.nlm.nih.gov.
[0092] Disclosed in U.S. Provisional Patent Application 61/523,167 filed August 12, 201 1 and International application PCT/US12/50389 filed August 10, 2012 (each of which is incorporated by reference herein, in its entirety), are selective 5PKC-modulatory peptides and compositions.
[0093] "Specific" or "specificity" refers to the selective modulation of bioactivity by a modulatory peptide or peptide composition. A modulatory peptide can be tested for its specificity of modulation (inhibiting or activating) by comparing the amount of a downstream effect, such as, for example (but not limited to) inhibition of binding of LACK or TRACK to its cognate binding partner in the presence or absence of the modulatory peptide or peptide composition. In one embodiment, the addition of a specific peptide inhibitor to a binding assay to measure binding of LACK or TRACK protein to its cognate binding protein in the presence and absence of the modulatory peptide results in a decrease in binding interaction between LACK or TRACK protein and its cognate binding protein. In some embodiments, the decrease in binding interaction between the LACK or TRACK protein and its cognate binding proteinin the presence of modulator peptide is at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, or at least a 100-fold greater than the decrease in binding interaction between LACK or TRACK protein and its cognate binding protein when the modulator peptide is not present.
II. Rational Design of LACK or TRACK Modulator Peptides
[0094] To identify inhibitors of protein-protein interactions between the RACK homologues (LACK and TRACK) and their cognate binding partners, the homology between RACK and these proteins was examined. LACK is highly homologous to the mammalian RACK1 (> 75% homology); however, there are regions of lower homology which may determine specific interactions of LACK with its binding partners. Five domains with low levels of amino acid conservation were identified. The non-conserved regions are mostly located in the outer most b-strand (b-strand-1 ) of each WD 40 domain. These regions were also conserved between different sub-species of Leishmania; since conserved sequences are usually indicative of an important function, it was hypothesized that peptides derived from these regions could be useful in targeting various Leishmania species without affecting the mammalian host. Thus, peptides were designed by identifying amino acid sequences found in LACK and TRACK proteins but not in RACK protein. Furthermore, sequences conserved in different Trypanosoma species were also identified.
[0095] Peptides derived from the non-conserved regions were then synthesized and tested towards their biological activity in Leishmania parasites. Because peptides typically do not cross membranes, a TAT-derived peptide, TAT47-57 (Chen, 2001 ) has been used to introduce PKC regulating-peptides into cells. Conjugating a carrier peptide to a cargo peptide is a well-established delivery technology shown to be effective in mammalian cells in culture, in animals, as well as in human subjects (Gray, 1997; Inagaki, 2003; Inagaki, 2005; Chen, 2001 ; Inagaki, 2003; Roe, 2007). The TAT47-57 delivery system has been successfully used to deliver an analogue of MARCKs-related protein effector domain (3DMRPED), a substrate for gp63 (Leishmania protease) to both Leishmania promastigotes and macrophages (Corradin, 2002). Furthermore, a study with Plasmodium employed a 10 amino-acid peptide to show that a single dose of 250 μΜ peptide inhibited 90% of parasite transformation (Dhawan, 2003 #1 1078). Thus, a similar system is used herein to deliver the peptides to the parasites and infected macrophages.
[0096] LACK or TRACK protein and its cognate binding protein may be found in different sub-cellular locations. Downstream effectors in the PKC pathway can include, but are not necessarily limited to, myristoylated alanine-rich C-kinase substrate (MARCKS) (Disatnik et al., 2002, J. Cell Sci., 1 15:2151-2163; Myat et al., 1997, Curr. Biol. 7:61 1 -614), occludin (Qi et al., 2008, J. Clin. Inv., 1 18:173-182), and several ion channels (Barman et al., 2004, Am. J. Physiol. Lung Cell. Mol. Physiol., 186:L1275- L1281 ) that are found at the plasma membrane; c-Abl is on the endoplasmic reticulum
(Qi et al., 2008, J. Cell Sci., 121 :804-813); dynamin-related protein 1 (Drp-1 ) on the mitochondria (Qi et al., 2010, Mol. Biol. Cell, 22:256-265); and pyruvate kinase and a heat shock protein (HSP27) are in the cytosol (Siwko et al., 2007, Int. J. Biochem. Cell Biol., 39:978-987).
[0097] Disclosed in U.S. Provisional Patent Application 61/523,167 filed August 12, 201 1 and International application PCT/US12/50389 filed August 10, 2012 (each of which is incorporated by reference herein, in its entirety), are selective 5PKC-modulatory peptides and compositions. Therein described is a rational design approach used to identify specific inhibitors of a single phosphorylation function of 5PKC-the phosphorylation of PDK. This rational approach also has been used to identify peptides that selectively inhibit PKC activity (Chen et al., Proc. Natl. Acad. Sci. U.S.A., 98:1 1 1 14- 1 1 1 19; Brandman et al., 2007, J. Biol. Chem., 282:41 13-4123) by interfering with PKC anchoring to its binding protein RACK (such as eV1-1 and 5V1 -1 ; FIG. 1 a, e) (Johnson et al., 1996, J. Biol. Chem., 24962-24966; Dorn et al., 1999, Proc. Natl. Acad. Sci. USA, 96:12798-12803; Brandman et al., 2007, J. Biol. Chem., 282:41 13-4123). Additionally, peptides that interfere with the auto-inhibitory interactions and thus act as activators of the corresponding isozyme (e.g., ΨεΡνΑΟΚ and ΨδΡνΑΰΚ) have been identified (Chen et al., Proc. Natl. Acad. Sci. USA, 98:1 1 1 14-1 1 1 19; Ron et al., 1995, Proc. Natl. Acad. Sci. USA, 92:492-496). The inhibitors therein described were then used to show that 5PKC- mediated phosphorylation of PDK is required for 5PKC-dependent cardiac injury following an ischemic event. One such inhibitor, ΨΡϋΚ, inhibited 5PKC-mediated phosphorylation of PDK, but not the phosphorylation of other 5PKC substrates, such as MARCKS or Drp1. Its specificity for 5PKC was also evident by the absence of ΨΡϋΚ effect in cells lacking 5PKC. ΨΡϋΚ peptide represents a short sequence of similarity between PDK2, a direct substrate of 5PKC, and 5PKC. Like the ΨΡϋΚ site, ALSTE, these peptides are all derived from the C2 domain. However, the action of ΨΡϋΚ is different.
[0098] In some embodiments, the modulatory peptide comprises a carrier moiety and a linker moiety. In some embodiments, the modulatory peptide is directly linked to a carrier moiety without a linker moiety. In some embodiments, the modulatory peptide does not comprise a linker moiety or a carrier moiety. In some embodiments, the peptide moiety comprises a carboxamide or cyclic peptide.
[0099] In some embodiments, a composition comprising a modulatory peptide will reduce a binding interaction between a LACK or TRACK protein to its cognate binding partnerbut will not affect the binding interaction between the cognate binding partner and any other of its non-LACK or non-TRACK binding partners. It is understood that the
effect of the modulatory peptide on the binding interaction between a LACK or TRACK protein to its cognate binding partner is merely to reduce the binding interaction between a LACK or TRACK protein and its cognate binding partner under equivalent assay conditions but in the absence of modulatory peptide. A "reduction" effect may encompass a decrease of 5%-20%, 10%-50%, 30%-50%, 40%-60%, 50%-80%, 70%- 90%, 80%-95%, or 90-99% in binding interaction between a LACK or TRACK protein and its cognate binding partner. Alternatively, this "reduction" effect encompasses at least a 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% decrease in binding interaction between a LACK or TRACK protein to its cognate binding partner.
[0100] The selective modulatory peptides herein disclosed can inhibit protein-protein interactions between LACK or TRACK protein and its cognate binding protein. The peptides can comprise a core amino acid sequence which is similar to the LACK- or TRACK-binding protein, substrate or cargo peptide identified as described herein. This core amino acid sequence is at least 60% identical to the LACK- or TRACK-binding protein, substrate or cargo sequence. However, a peptide which can selectively inhibit interaction of the LACK or TRACK with its cognate binding partner may be significantly longer than this core sequence of 5 amino acid residues. For example, a selective modulator peptide may be 5-20 amino acids in length, or 6-15 amino acids in length. In some embodiments, the additional amino acids, which may be N-terminal, C-terminal or both N-terminal and C-terminal to the core sequence are derived from a LACK- or TRACK-binding protein, substrate or cargo protein sequence. Accordingly, the entire length of the selective modulator peptide will be at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% identical to a same length of sequence within the LACK- or TRACK-binding protein, substrate or cargo peptide for which the modulator peptide is effective. Although the peptides are described primarily with reference to amino acid sequences from Homo sapiens, it is understood that the peptides are not limited to the specific amino acid sequences set forth herein.
[0101] It will be appreciated that the selective modulatory peptides can be used in native form or modified by conjugation to a carrier, such as those described below. Alternatively, one or two amino acids from the sequences can be substituted or deleted and exemplary modifications and derivatives and fragments for each peptide are given below.
III. Peptide synthesis
[0102] All commercially available solvents and reagents were used without further purification, dichloromethane (DCM), N-methyl-2-pyrrolidone (NMP), triisopropylsilane
(TIS), Ν,Ν-diisopropylethylamine (DIEA), 0-benzotriazole-N,N,N',N'-tetramethyl- uronium-hexafluoro-phosphate (HBTU), 1-Hhydroxybenzotriazole (HOBt) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich Chemicals (MO, USA); dimethylformamide (DMF) (Alfa Aesar, MA, USA); piperidine (Anaspec, CA, USA) rink amide AM resin (substitution 0.49 mmol/g, CBL Biopharma LLC, CO, USA), Fmoc- protected amino acids were obtained from Advanced ChemTech and GL Biochem (KY, USA and Shanghai, China). Side chains of the amino acids used in the synthesis were protected as follows: Boc (Lys/Trp), But (Ser/Thr/Tyr), OBut (Asp/Glu), Pbf (Arg) and Trt (Asn/Cys/Gln/His). Peptides were chemically synthesized using Liberty Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC, USA) with an additional module of Discover (CEM Corporation, Matthews, NC, USA) equipped with fiber-optic temperature probe for controlling the microwave power delivery following the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy in a 30 ml teflon reaction vessel. Deprotection was performed with a 20% piperidine in DMF with 0.1 M HOBt solution. Coupling reactions were performed with HBTU in DMF (0.1 1 M), amino acids in DMF (0.12 M) and DIEA in NMP solution (0.25 M). Each deprotection and coupling reaction was performed with microwave energy and nitrogen bubbling. Microwave cycle included two deprotection steps of 30 sec and 180 sec, each. All coupling reactions lasted 300 sec. Peptide cleavage from the resin and deprotection of the amino acids side chains were carried out with TFA/TIS/ H20 /phenol solution (90:2.5:2.5:5 v/v/v/v) for 4 h at room temperature. The resin was washed with TFA. The crude products were precipitated with diethyl ether, collected by centrifugation, dissolved in H20/ CH3CN and lyophilized. Products were analyzed by analytical RP-HPLC (Shimadzu LC-20 equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 χ LC- 20AD solvent delivery unit, SIL-20AC autosampler, DGU-20A5 degasser from Shimadzu, MD, USA) using an ultro 120 5pm C18Q (4.6 mm ID 150 mm) column (Peeke Scientific, CA, USA) at 1 mL/min. The solvent systems used were A (H20 with 0.1 % TFA) and B (CH3CN with 0.1 % TFA). For separation, a linear gradient of 5-95% B in 45 min was applied and the detection was at 215 nm.
[0103] Products were purified by preparative reverse-phase high-pressure liquid chromatography (RP-HPLC; Shimadzu LC-20) equipped with CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 χ LC-6AD solvent delivery unit and FRC-1 OA fraction collector from Shimadzu, MD, USA), using an XBridge Prep OBD C18 5pm (19 mm x 150 mm) column (Waters, MA, USA) at 10 mL/min. The solvent systems used were A (H20 with 0.1 % TFA) and B (CH3CN with 0.1 % TFA). For
separation, a linear gradient of 5-95% B in 45 min was applied and the detection was at 215 nm.
[0104] Peptides were conjugated to TAT47-57 carrier peptide through an amide bond as part of the synthesis with different spacers.
III. Modulatory Peptide Compositions Comprising a Carrier Moiety
[0105] The modulatory peptide useful in inhibiting LACK or TRACK protein-protein interactions with its cognate binding protein can be attached or linked to a peptide moiety which facilitates transfer of the modulatory peptide composition across a cell membrane. This peptide carrier may be any one of a number of peptide carriers known in the art for facilitating transfer across cell membranes, including Tat, the Drosophila Antennapedia protein, a polycationic peptide such as polyarginine or polylysine) (e.g., (R)7), penetratin, Tat, VT5, MAP, Transportan, Transportan-10, pVEC, pISL, Pep-1 , and Mouse PrPC (1-28) (see Lundberg et al., 2003, J. Mol. Recognit, 16:227-233, U.S. Pat. Pub. Nos. 2003/0104622 and 2003/0199677). In some embodiments, the carrier peptide is Tat-derived transport polypeptide (U.S. Pat. Nos. 5,747,647 and 5,804,604; Vives, et al. J. Biol. Chem., 272:16010-16017 (1997)), polyarginine (U.S. Pat. Nos. 4,847,240 and 6,593,292; Mitchell et al., 2000; Rothbard et al., 2002) or Antennapedia peptide (U.S. Pat. No. 5,888,762). The disclosures of these references are incorporated herein in their entirety.
[0106] The modulatory peptide may be linked to the carrier peptide by a disulfide bond. In some embodiments, the disulfide bond is formed between two cysteines, two cysteine analogs or a cysteine and a cysteine analog. In this embodiment, both the modulatory peptide and the carrier peptide contain at least one cysteine or cysteine analog. The cysteine residue or analog may be present as the N-terminal or C-terminal residue of the peptide or as an internal residue of the modulatory peptide and of the carrier peptide. The disulfide linkage is then formed between the sulfur residues on each of the cysteine residues or analogs. Thus, the disulfide linkage may form between, for example, the N-terminus of the modulatory peptide and the N-terminus of the carrier peptide, the C-terminus of the modulatory peptide and the C-terminus of the carrier peptide, the N-terminus of the modulatory peptide and the C-terminus of the carrier peptide, the C-terminus of the modulatory peptide and the N-terminus of the carrier peptide, or any other such combination including at any internal position within the modulatory peptide and/or the carrier peptide.
[0107] The modulatory peptide can alternatively be part of a fusion protein. Typically, to form a fusion protein, the peptide is bound to another peptide by a bond other than a
Cys-Cys bond. An amide bond from the C-terminal of one peptide to the N-terminal of the other is exemplary of a bond in a fusion protein. This embodiment encompasses the presence of a peptide bond between and linking the modulatory and carrier peptides to form a single linear peptide composition comprising both the modulatory peptide and the carrier peptide. The modulatory peptide may be N-terminal to the carrier peptide, or the carrier peptide may be N-terminal to the modulatory peptide.
[0108] A short linker peptide may be present between the modulatory peptide and the carrier peptide within the single linear peptide composition. The linker peptide may comprise 1 to 15 amino acids. Alternatively, the linker peptide may comprise 2 to 10 amino acids, 3 to 10 amino acids, 4 to 10 amino acids, 2 to 8 amino acids, 3 to 7 amino acids, or 4 to 6 amino acids. The linker peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids. In some embodiments, the linker peptide comprises
1 , 2, 3, 4, or 5 glycine residues. In some embodiments, the linker peptide comprises 1 ,
2, 3, 4 or 5 alanine residues. In some embodiments, the methods known as "alanine scanning" mutagenesis or "deletion scanning" are used to determine the significance, function or utility of particular amino acid residues in a peptide. In some embodiments, the linker peptide comprises at least 1 serine residue. In some embodiments, the linker is Gly-Ser-Gly. It is understood that the linker peptide may comprise any amino acid or amino acid analog, The single linear peptide composition may alternative have a single amino acid present between the modulatory peptide and the carrier peptide.
[0109] Kinase modulators are very important for basic research as well as drugs. Numerous kinase modulators have been developed earlier. Most of these regulators are small molecules many with broad activity and other with higher selectivity (Karaman et al., 2008, Nat. Biotech., 26:127-132). That a modulator peptide can be specific for a single signaling molecule is surprising and novel. Such specific regulators can be rationally designed and these peptides provide missing tools to determine the role of one of several cellular functions of, for example, a given PKC isozyme. This approach is likely applicable to other signaling proteins, allowing the generation of separation-of function regulators of other protein-protein interactions.
IV. Methods of Use
[0110] The modulatory peptides and peptide compositions described herein may be administered to a subject in need thereof to prevent or reduce infection by a parasite, e.g., Leishmania or Trypanosoma species. Such peptides are useful for slowing or inhibiting the progression of parasitic infection in a subject, such that the subject more closely resembles a healthy animal.
[0111] In certain embodiments, there is provided a method of treating an individual at risk or with an established parasitic infection. Such a method comprises the step of administering to the individual a pharmacologically effective amount of a modulatory peptide or composition that selectively inhibits LACK or TRACK protein-protein interactions with its cognate binding protein. "An effective amount" or "pharmacologically effective amount" refers to the amount of peptide or composition required to confer a therapeutic effect on the treated subject, e.g., reduced susceptibility to parasitic infection. Effective doses will also vary, as recognized by those skilled in the art, depending on the route of administration, the excipient usage, and the optional co-usage with other therapeutic treatments. In still yet another embodiment, there is a method of protecting the subject from parasitic infection. Such a method comprises administering a modulatory peptide composition wherein the administering results in increased protection of macrophages from parasitic infection, reduces apoptotic cell death caused by parasitic infection, as compared to said results in the absence of administering a modulatory peptide composition or administering a control peptide composition. The reducing can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease, or any value or range there between, in the amount of disease due to parasitic infection, including but not limited to lesion size.
[0112] In certain embodiments, modulatory peptides and peptide compositions may be co-administered in a composition with a second therapeutic agent. In this manner, one skilled in the art will recognize that the modulatory peptides individually, in combination, or combined with a second therapeutic agent, may be used to prepare a medicament for the slowing or inhibiting the progression of, for example, parasitic infection, leishmaniasis, trypanosomiasis, or Chagas disease.
V. Formulations
[0113] A pharmaceutical composition comprising a described compound and at least one pharmaceutically acceptable excipient or carrier is provided. Methods of preparing such pharmaceutical compositions typically comprise the step of bringing into association a described compound with or without a carrier moiety and, optionally, one or more accessory ingredients. The described compounds and/or pharmaceutical compositions comprising same may be formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. Typically, formulations are prepared by uniformly and intimately bringing into association a described compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Pharmaceutical compositions of the present
invention suitable for parenteral administration comprise one or more described compounds in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, amino acids, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
[0114] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
[0115] These pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the described compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include agents to control tonicity, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
[0116] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
[0117] For example, a described compound may be delivered to a human in a form of solution that is made by reconstituting a solid form of the drug with liquid. This solution may be further diluted with infusion fluid such as water for injection, 0.9% sodium chloride injection, 5% dextrose injection and lactated ringer's injection. The reconstituted and diluted solutions may be used within 4-6 hours for delivery of maximum potency.
Alternatively, a described compound may be delivered to a human in a form of tablet or capsule.
[0118] Injectable depot forms are made by forming microencapsulated matrices of the described compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
[0119] When the described compounds are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition may contain 0.2-25%, preferably 0.5-5% or 0.5-2%, of active ingredient. These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including, e.g., subcutaneous injection, subcutaneous depot, intravenous injection, intravenous or subcutaneous infusion. These compounds may be administered rapidly (within <1 minute) as a bolus or more slowly over an extended period of time (over several minutes, hours or days). These compounds may be delivered daily or over multiple days, continuously or intermittently. In one embodiment, the compounds may be administered transdermally (e.g., using a patch, microneedles, micropores, ointment, microjet or nanojet).
[0120] Regardless of the route of administration selected, the described compounds, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
[0121] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
[0122] The selected dosage level will depend upon a variety of factors including the activity of the particular described compound employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition,
general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
[0123] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the described compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
[0124] In general, a suitable daily dose of a described compound will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intramuscular, transdermal, intracerebroventricular and subcutaneous doses of the described compounds for a patient, when used for the indicated effects, will range from about 1 μg to about 5 mg per kilogram of body weight per hour. In other embodiments, the dose will range from about 5 μg to about 2.5 mg per kilogram of body weight per hour. In further embodiments, the dose will range from about 5 μg to about 1 mg per kilogram of body weight per hour.
[0125] If desired, the effective daily dose of a described compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In one embodiment, the described compound is administered as one dose per day. In further embodiments, the compound is administered continuously, as through intravenous or other routes. In other embodiments, the compound is administered less frequently than daily, such as every 2-3 days, in conjunction with dialysis treatment, weekly or less frequently.
[0126] The subject receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
[0127] The described compounds may be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
VI. Routes of Administration for Disclosed Compounds
[0128] These compounds may be administered to humans and other animals for therapy by any suitable route of administration. As used herein, the term "route" of administration is intended to include, but is not limited to subcutaneous injection, subcutaneous depot, intravenous injection, intravenous or subcutaneous infusion, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intraadiposal administration, intraarticular administration, intrathecal administration, epidural administration, inhalation, intranasal administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisternal administration and topical administration, transdermal administration, or administration via local delivery (for example by catheter or stent).
[0129] Transdermal drug delivery to the body is a desirable and convenient method for systemic delivery of biologically active substances to a subject, and in particular for delivery of substances that have poor oral bioavailability, such as proteins and peptides. The transdermal route of delivery has been particularly successful with small (e.g., less than about 1 ,000 Daltons) lipophilic compounds, such as scopolamine and nicotine, that can penetrate the stratum corneum outer layer of the skin, which serves as an effective barrier to entry of substances into the body. Below the stratum corneum is the viable epidermis, which contains no blood vessels, but has some nerves. Deeper still is the dermis, which contains blood vessels, lymphatics and nerves. Drugs that cross the stratum corneum barrier can generally diffuse to the capillaries in the dermis for absorption and systemic distribution.
[0130] Technological advances in transdermal delivery have focused on addressing the need in the art to deliver hydrophilic, high molecular weight compounds, such as proteins and peptides, across the skin. One approach involves disruption of the stratum corneum using chemical or physical methods to reduce the barrier posed by the stratum corneum. Skin microporation technology, which involves the creation of micron dimension transport pathways (micropores) in the skin (in particular, the micropores in the stratum corneum) using a minimally invasive technique, is a more recent approach. Techniques to create micropores in the skin (stratum corneum) include thermal microporation or ablation, microneedle arrays, phonophoresis, laser ablation and radiofrequency ablation (Prausnitz and Langer (2008) Nat. Biotech. 1 1 :1261 -68; Arora et al., Int. J. Pharmaceutics, 364:227 (2008); Nanda et al. Current Drug Delivery, 3:233 (2006); Meidan et al. American J. Therapeutics, 1 1 :312 (2004)).
[0131] In one embodiment, the modulator peptide is delivered via microporation. Any one of a number of techniques for microporation is contemplated, and several are briefly described.
[0132] Microporation can be achieved by mechanical means and/or external driving forces, to breach the stratum corneum to deliver the calcimimetic agents described herein through the surface of the skin and into the underlying skin layers and/or the bloodstream.
[0133] In some embodiments, the microporation technique is ablation of the stratum corneum in a specific region of the skin using a pulsed laser light of wavelength, pulse length, pulse energy, pulse number, and pulse repetition rate sufficient to ablate the stratum corneum without significantly damaging the underlying epidermis. The calcimimetic agent is then applied to the region of ablation. Another laser ablation microporation technique, referred to as laser-induced stress waves (LISW), involves broadband, unipolar and compressible waves generated by high-power pulsed lasers. The LISWs interact with tissues to disrupt the lipids in the stratum corneum, creating intercellular channels transiently within the stratum corneum. These channel, or micropores, in the stratum corneum permit entry of the calcimimetic agent.
[0134] Sonophoresis or phonophoresis is another microporation technique that uses ultrasound energy. Ultrasound is a sound wave possessing frequencies above 20 KHz. Ultrasound can be applied either continuously or pulsed, and applied at various frequency and intensity ranges (Nanda et a/., Current Drug Delivery, 3:233 (2006)).
[0135] Another microporation technique involves the use of a microneedle array. The array of microneedles when applied to a skin region on a subject pierce the stratum corneum and do not penetrate to a depth that significantly stimulates nerves or punctures capillaries. The patient, thus, feels no or minimal discomfort or pain upon application of the microneedle array for generation of micropores through which the modulatory agent is delivered.
[0136] Microneedle arrays comprised of hollow or solid microneedles are contemplated, where the modulatory agent can be coated on the external surface of the needles or dispensed from the interior of hollow needles. Examples of microneedle arrays are described, for example, in Nanda et a/., Current Drug Delivery, 3:233 (2006) and Meidan et al. American J. Therapeutics, 1 1 :312 (2004). First generation microneedle arrays were comprised of solid, silicon microneedles that were externally coated with a therapeutic agent. When the microarray of needles was pressed against the skin and removed after about 10 seconds, the permeation of the agent on the needles into the body was readily achieved. Second generation microneedle arrays were
comprised of microneedles of solid or hollow silicon, polycarbonate, titanium or other suitable polymer and coated or filled with a solution of the therapeutic compound. Newer generations of microneedle arrays are prepared from biodegradable polymers, where the tips of the needles coated with a therapeutic agent remain in the stratum corneum and slowly dissolve.
[0137] The microneedles can be constructed from a variety of materials, including metals, ceramics, semiconductors, organics, polymers, and composites. Exemplary materials of construction include pharmaceutical grade stainless steel, gold, titanium, nickel, iron, tin, chromium, copper, palladium, platinum, alloys of these or other metals, silicon, silicon dioxide, and polymers. Representative biodegradable polymers include polymers of hydroxy acids such as lactic acid and glycolic acid polylactide, polyglycolide, polylactide-co-glycolide, and copolymers with poly(ethylene glycol), polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide- co-caprolactone). Representative non-biodegradable polymers include polycarbonate, polyester, and polyacrylamides.
[0138] The microneedles can have straight or tapered shafts. In one embodiment, the diameter of the microneedle is greatest at the base end of the microneedle and tapers to a point at the end distal the base. The microneedle can also be fabricated to have a shaft that includes both a straight (untapered) portion and a tapered portion. The needles may also not have a tapered end at all, i.e. they may simply be cylinders with blunt or flat tips. A hollow microneedle that has a substantially uniform diameter, but which does not taper to a point, is referred to herein as a "microtube." As used herein, the term "microneedle" includes both microtubes and tapered needles unless otherwise indicated.
[0139] Electroporation is another technique for creating micropores in the skin. This approach uses the application of microsecond or millisecond long high-voltage electrical pulses to created transient, permeable pores within the stratum corneum.
[0140] Other microporation techniques include use of radio waves to create microchannels in the skin. Thermal ablation is yet another approach to achieve delivery of larger molecular weight compounds transdermally.
[0141] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties. However, where a patent, patent application, or publication containing express definitions is incorporated by reference, those express definitions should be understood to apply to the incorporated patent, patent application, or publication in which they are found, and not necessarily to the text
of this application, in particular the claims of this application, in which instance, the definitions provided herein are meant to supersede.
[0142] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to implement the disclosed subject matter, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some minor errors and deviations may have been introduced. Unless indicated otherwise, parts are parts by weight, temperature is in °C and pressure is at or near atmospheric.
EXAMPLES
[0143] The following examples are illustrative in nature and are in no way intended to be limiting.
EXAMPLE 1
RATIONAL DESIGN OF INHIBITORY PEPTIDES
[0144] Conservation through evolution is one of the criteria established for the identifying sequences important for protein-protein interactions (Souroujon et a/., 1998, Nat. Biotechnol., 16:919-924; Qvit et a/., 2010, Drug Disc. Today: Dis. Mech., 7:e87- e93).
[0145] Interfering peptides able to disrupt protein-protein interaction between homologs of RACK and C2-containing proteins in the parasites were predicted to be able to inhibit key biological functions and therefore to provide novel therapeutic agents for diseases associated with infections with these parasites. We used a rational approach to design the peptides, in a manner similar to those previously published [{Souroujon, 1998 #24}; {Dorn, 1999 #18}; {Chen, 2001 #25}; {Qvit, 2010 #9082}]. We synthesized 20 peptides using solid phase peptide synthesis method.
[0146] Peptide synthesis. Peptides were synthesized by conventional manual synthesis, or using Microwave by Liberty Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC, USA) or by American Peptide (CA, USA). In some embodiments, peptides were conjugated to a TAT carrier (see below) by disulfide bond as described in Chen et al. (2001 , Chem. Biol., 8:1 123-1 129). In some embodiments, no disulfide bridge is used, but rather, peptides are synthesized and connected to TAT as one polypeptide:
N-terminus - TAT - spacer - cargo - C-terminus
[0147] In some embodiments, the cargo is at the N terminus and TAT is at the C- terminus. The C-terminus of the peptides was modified to C(0)-NH2 using Rink Amide
AM resin to increase stability (as described in Sabatino et al. {Cur. Opin. in Drug Disc. & Dev., 1 1 :762-770). Peptides were analyzed by analytical reverse-phase high-pressure liquid chromatography (RP-HPLC) (Shimadzu, MD, USA) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and purified by preparative RP- HPLC. These peptides are linked to a peptide carrier, TAT, also known as Tat.47.57 (RRRQRRKKRGY; hereinafter SEQ ID NO: 1 ) to enable their transmembranal delivery. The TAT peptide may also be used in the reverse orientation, (YGRKKRRQRRR; hereinafter SEQ ID NO: 2). Peptide 213 was synthesized as negative control and was not active. Two Glycines ("GG"), a Glycine-Serine-Glycine tripeptide ("GSG"), a beta- Alanine ("b-Ala"), gamma-amino-n-butyric acid (also known as "GABA" or "Gaba"), or Fmoc-Epsilon-Ahx-OH (CAS: 88574-06-5) were used as spacers (linkers). In some embodiments, no spacer was used. In some embodiments, cyclic peptides without TAT are used (See Figure 1 , for example). In some embodiments, TAT may not be required in order for the peptide to cross cell membranes.
[0148] Exemplary combinations of carrier, spacer and cargo in the modulatory peptides are shown below:
Peptide# Carrier, Tat Spacer Cargo
007 RRRQRRKKRGY (SEQ ID NO 1 ) Gaba CVSLAHATD (SEQ ID NO: 3 )
0 91 RRRQRRKKRGY (SEQ ID NO 1 ) GG CVSLAHATD (SEQ ID NO: 3 )
212 RRRQRRKKRGY (SEQ ID NO 1 ) b-Ala CVSLAHATD (SEQ ID NO: 3 )
213 (no Tat) (no spacer) CVSLAHATD (SEQ ID NO: 3 )
218 CVSLAHATD (SEQ ID NO: 3 ) (cargo) Gaba RRRQRRKKRGY (SEQ ID NO: 1 ) ,Tat)
21 9 RRRQRRKKRGY (SEQ ID NO 1 ) (no spacer) CVSLAHATD (SEQ ID NO: 3 )
008 RRRQRRKKRGY (SEQ ID NO 1 ) Gaba RNGQCQRK (SEQ ID NO: 5 )
0 92 RRRQRRKKRGY (SEQ ID NO 1 ) GG RNGQCQRK (SEQ ID NO: 5 )
225 RRRQRRKKRGY (SEQ ID NO 1 ) GSG HEFLRD (SEQ ID NO: 6 )
228 RRRQRRKKRGY (SEQ ID NO 1 ) GSG NVIRVWN (SEQ ID NO: 7 )
0 93 RRRQRRKKRGY (SEQ ID NO 1 ) GG VNGGKCERTLK (SEQ ID NO: 8 )
22 6 RRRQRRKKRGY (SEQ ID NO 1 ) GSG STGEQLFKINVESP (SEQ ID NO: 9 )
0 94 RRRQRRKKRGY (SEQ ID NO 1 ) GG TPDGAKPSE (SEQ ID NO: 1 0 )
230 RRRQRRKKRGY (SEQ ID NO 1 ) GSG RSLSVYD (SEQ ID NO: 11 )
0 95 RRRQRRKKRGY (SEQ ID NO 1 ) GG QKKGDI TDPYVKL (SEQ ID NO: 12 )
231 RRRQRRKKRGY (SEQ ID NO 1 ) GSG QKKGDI TDPYVKL (SEQ ID NO: 12 )
012 RRRQRRKKRGY (SEQ ID NO 1 ) Gaba KTSWRNN (SEQ ID NO: 13 )
0 96 RRRQRRKKRGY (SEQ ID NO 1 ) GG KTSWRNN (SEQ ID NO: 13 )
0 97 RRRQRRKKRGY (SEQ ID NO 1 ) GG GLNPYW ET (SEQ ID NO: 14 )
232 RRRQRRKKRGY (SEQ ID NO 1 ) GSG GLNPYW ET (SEQ ID NO: 14 )
EXAMPLE 2
EFFECTS OF INHIBITORY PEPTIDES IN BIOLOGICAL SYSTEMS
Leishmania
Peptide treatment on Leishmania promastigote cultures
[0149] The bioactivity of the peptides on parasite viability of Leishmania amazonensis (New World Leishmania) and Leishmania donovani (Old World Leishmania) promastigotes was assessed.
Leishmania viability in culture
[0150] Cultures of different strains of parasites (causative of the 3 different clinical forms: L. major- cutaneous, L. donovani- visceral and L. braz/V/ens/s-muco-cutaneous), carried out in triplicates, were treated with different concentrations of peptides, using two protocols (1 ) a single dose at time 0 for one hour followed by 24 hrs of culturing or (2) parasites were treated with three treatments every 4 hours during the first hours of the study. The parasites were incubated with increasing concentrations (0-243 μΜ) of various peptides, and parasite proliferation and viability were evaluated by MTT assay after 2, 24, 48 and 72 hours (Sereno, 1997). Peptides had a marginal effect at very low concentration (3 μΜ), while they were almost 100% effective at moderate concentrations (81 μΜ, peptide LLM-1 .4) and peptide (LLM-2.1 ) induced almost complete parasite cell death at a higher concentration (243 μΜ). The IC50 of the most active peptides is considered low for a first screen for a lead compound.
Trypanosoma cruzi
[0151] Some of the non-conserved regions in LACK are conserved in TRACK; for example, there is 75.0% identity between LACK and TRACK in domain ll2 and much lower homology in this domain is found between RACK and LACK or RACK and TRACK. Thus, the peptides were tested for activity against T. cruzi trypomastigote forms of the parasite infective for mammalian cells and also found in the blood of infected mammals. Peptides l , ll2 and \ dramatically decreased the motility and the number of intact parasites, and according to the screening test, some peptides were able to diminish parasite viability at a concentration of 100 μΜ. In general, peptides with the same sequence but with different linkers displayed comparable bioactivity. Peptide LLM- 1.6 lacking TAT47-57 did not show any effect on T. cruzi trypomastigotes indicating that (as in the case with Leishmania) peptides must enter the parasite in order to be effective. TAT47-57 peptide lone had no effect on parasite viability.
[0152] Peptides LLM-1.1 and LLM-2.2 were also tested in dose-response studies, and these peptides displayed higher trypanocidal activity. A strong correlation between concentration and trypanocidal activity was shown for these peptides.
Trypanosoma viability in culture
[0153] Trypanocidal activity of the peptides was tested against trypomastigotes forms. Trypomastigotes Y stain (2 x 106), obtained from 5° or 6° day of infection of cultures of LLC-MK2 cell line (Andrews, 1982). The assay was carried out in fresh modified Eagle's medium (MEM) phenol red free with 2% FBS and incubated with different peptides at 100 μΜ for two hours at 37 °C and 5% C02. The disruption of parasite's motility was monitored by microscopic examination. Parasite viability was measured by a colorimetric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-2H-tetrazolium monosodium salt (WST-1 ) reagent (Roche). Cell viability was determined by the level of reduced WST-1 measured at 450 nm, expressed as a percentage of the control as a reference.
[0154] Peptides IC5o assay: The peptides with better trypanocidal activity (LLM-1.1 and LL||-2.2) were selected to IC5o assay. The trypomastigotas (2 x 106) were treated with the selected peptides at different concentrations (0, 5, 10, 25, 50, 75 and 100 μΜ) for 2 hours at the same conditions previously described.
Peptide treatment on parasite in culture
[0155] Parasites were treated with a single dose (100 μΜ) of peptide for two hours.
[0156] Peptides derived from LACK or TRACK protein and homologous to regions in Trypanosoma cruzi (TcRACKI ) linked to Tat carrier peptide effectively killed trypomastigote forms of Trypanosoma cruzi in a dose responsive manner within one hour of treatment, independent of the linker. Peptide alone, without Tat, had no effect.
[0157] Assay of the inhibition of Leishmania donovani 1S2D promastigote by LACK / TRACK modulatory peptides:
93 98.66
226 95.49
336 92.60
337 88.76
338 96.47
342 93.10
305 98.70
339 92.46
227 56.88
343 64.35
228 84.02
344 82.93
229 79.50
230 97.44
345 2.03
346 100.44
95 97.82
231 96.99
96 84.78
232 80.98
309 71 .03
330 96.05
258 96.54
Positive (218) 99.00
Negative (213) 101.50
[0158] In this in vitro data, the lower the number, the higher the activity of the modulatory peptide; likewise, the higher the number, the lower the activity. Thus, 100% would be a completely inactive peptide. For example, upon treatment with modulatory peptides 340d, 341a, 341 c, 341 and 345, the average parasite viability was in the 0-25% range, indicating that these peptides were highly active. For example, it was noted that after 48 hours, incubation with peptide number 345, the L. donovani promastigotes were nearly all dead. Modulatory peptides 340e, 308, 329, 331 , 341 b and 341 d reduced parasite viability to 25-50%, and modulatory peptides 227, 343 and 309 reduced parasite viability to 50-75% of parasite viability absent treatment with modulatory peptide.
[0159] Using a variety of assays, we found that treatment with the peptides:
[0160] Several modulatory peptides had high levels of biological activity, i.e., were found to be effective in decreasing parasite viability against different strains of Leishmania (including both Old and New world strains) as well as Trypanosoma cruzi, and pre-treatment of Leishmania parasites with these peptides reduced parasite infection of macrophages by Leishmania and Trypanosoma parasites.
[0100] A structure activity relationship study based on these lead compounds was performed, and an increase in peptide activity against the parasite reducing the number of viable free forms and intracellular parasites was observed. Certain parasite proteins (amongst them a previously described TRACK binding protein) bound to a biologically active peptide, confirming that the rationally designed peptides may compete with TRACK for protein binding.
[0161] An additional assay was performed, in which peptides were added every 4 hours, and parasite viability assessed after 24 h. Peptides derived from domains lh, 112 and \ induced cell death at IC50 between 9-33 μΜ. Other LACK-derived modulatory peptides (e.g., peptides derived from domain IVi) had a modest effect, whereas peptides derived from domain ll had marginal effects. Because a peptide derived from domain lh had a significant effect in all assays, this peptide was chosen for optimization in structure activity relationship (SAR) studies. The TAT peptide was used to deliver the peptides into the cell, and various linkers between the cargo and the TAT peptide were employed to test and optimize linker length. Addition of two Gly amino acids to the linker between peptide LLM-1.1 and the carrier peptide (GG, LLM-1.1 ) was found to be more effective against Leishmania amazonensis promastigotes compared to peptides with one amino acid spacer such as, beta-alanine (LLn-1.2), gamma-aminobutyric acid (Gaba) (LL||-1.4) or with no spacer at all (LLM-1.3). The location of the TAT carrier peptide was not important as was observed by comparing peptides LLM-1.4 in which the TAT sequence is at the N-terminus and LLM-1 .5 at the C-terminus. Moreover, peptide lacking
TAT but containing the same cargo sequence (LLM-1.6) was found to have no cytotoxic effect at all, demonstrating the utility of TAT for cell delivery.
Peptide treatment on infected macrophages
[0162] Next, the peptides were assessed for efficacy in reducing the ability of promastigotes to infect macrophages. Promastigotes of L. amazonensis were incubated with peptides (0-200 μΜ) for 1 hour, parasites were then washed and used to infect macrophages. Infected cells were incubated for an additional 30 h without any further treatment. Peptide LLM-1.4 was the most effective peptide in this assay, resulting in over 75% reduction in the number of infected cells at 200 μΜ. Although, the concentration for the peptides is somewhat high, promastigotes were treated only once prior to infection which was assessed after 30 h. Therefore, peptides LLM-1.4 and LLM-2.1 had an effect not only on parasite viability but also on their ability to infect macrophages and to propagate in infected host cells, characteristics of great interest for development of therapeutic peptides.
Assessment of Intracellular parasite load of infected macrophages
[0163] Peritoneal macrophages from BALB/c mice (susceptible to Leishmania infection) or a murine macrophage cell line, J774, adhered onto coverslips (Callahan, 1997) were infected with Leishmania parasites at a 1 :10 (macrophage to parasite ratio) at 34°C in a humidified environment containing 5% C02. Where indicated, promastigotes were treated with the indicated peptides XX minutes prior to infection and cultures were maintained for an additional 96 h. Cells were fixed after the infection and stained with Giemsa for microscopical estimation of amastigote infection-rate/survival. The number of infected macrophages and the number of parasites per macrophage were determined.
Cell viability assay in culture:
[0164] Cardiac myocytes isolated from 1 -day-old Sprague-Dawley rat litters were treated with modulatory peptides for 48 hours. The cell viability was measured using an in vitro toxicology assay MTT-based kit (Sigma), according to the manufacturer's instruction. As a positive control, cardiac myocytes were treated with H202 (100 μΜ) for 24 hours.
Cardiac cells assay in culture, determining the effect of Leishmania-der'wed peptides on PKCbll function:
[0165] Cardiac myocytes were treated with 20 μΜ peptides for 20 min followed by 10 nM PMA as indicated. Cells were lysed and the lysate was centrifuged at 800 g and then the nuclei discarded and the supernatant then spun at 100,000 g to obtain the particulate fraction in which the level of beta2 PKC was determined. The level of beta2 at the
membrane is a marker of activation and translocation and dependent on the interaction of beta2 with RACK1. By WB analysis, the level of beta2 PKC at the membrane in the presence of the peptides was determined. Beta V5-3 peptide, a known beta2 specific inhibitor, was used as a control. Note the increase of translocation of PKC in the presence of PMA with no peptide present of 2 fold.
Primary cardiacmvocvtes, immunoprecipitation and kinase assay:
[0166] Neonatal cardiac myocytes were isolated from neonatal rats as previously described (Qi, 2007). Cardiac myocytes were isolated by using the cardiomyocyte isolation kit from Cellutron. Cardiac myocytes represent 90-95% of total adherent cells. Cells were maintained in MEM Eagle's with Earle's BSS media (containing 50 U/ml penicillin, 80 pmol/l vitamin B12, 0.1 mM bromodeoxyuridine (BrdU) and 80 pmol/l vitamin C) with 10% serum. After 4 days, the cells were treated with respective peptides at 1 mM for 15 min before adding 10 nM PMA for 30 min.
[0167] After treatment cardiac myocytes were washed with cold phosphate-buffered saline, scraped in homogenization buffer (Tris-HCI 50 mM, EGTA 1 mM, EDTA 1 mM and 250 mM sucrose), and lysed by passing the cell extract though a 26 G needle. The extract was spun at 100,000 x g for 30 min at 4 °C. The supernatants correspond to the cytosolic fraction. The pellets were resuspended in homogenization buffer containing 0.1 % Triton X-100 and correspond to the particulate fractions.
[0168] ΡΚΟβΙΙ was immunoprecipitated from the particulate fractions as described previously (Disatnik, 2002) using anti-PKCpil antibody (Santa Cruz Biotechnology). The immunoprecipitated kinase of respective peptide treatment was incubated 30 min at 37 °C in 40 μΙ of binding buffer (20 mM Tris-HCI, 20 mM MgCI2, 1 mM DTT, 25 mM ATP) containing 5 pCi [γ32Ρ] ATP (4500 Ci/mmole, ICN) and 10 mM MBP. After incubation, assays were terminated by adding loading buffer containing 5% SDS. The samples were loaded on 14% SDS acrylamide gel and transferred to nitrocellulose. The level of phosphorylated MBP was analysed by exposing the nitrocellulose to autoradiography.
[0169] In some embodiments, the reaction was performed in presence of PKC activators, phosphatidylserine (60 pg/ml) and sn-1 ,2 dioleoylglycerol (2 pg/ml). 460 ul phophatidylserine and 16 μΙ DG were added together and evaporated using nitrogen or argon. The mixture was dried and 4 ml Tris-Hcl 20 mM added, then the mixture was sonicated for three times at medium force. This preparation may be used for one month when stored at 4 °C.
Cell viability assay
[0170] LLC-MK2 cells were seeded in 96-well plates at a density of 8,000 cells/well 24 h prior to treatments. All cell treatments were carried out in MEM medium phenol red- free supplemented with 2% FBS. The cells received three doses of 100 μΜ of each peptideo (LLII-1.1 , LLII-2.2) at time-point 0, 3 and 6 h, 12 h after the last addition of peptide, cell viability was determined using WST-1 (Roche, Indianapolis, IN) according to the manufacturer's protocol. Cell morphology was accessed by light microscopy.
[0101]
Peptide treatment on post macrophage infection
[0171] Macrophages were infected with promastigotes for 2 hours, cells were washed and then treated with peptides for 48 h, with changes in medium every 12 h.
[0172] 100 μΜ of peptide was added to the L. donovani (Old World Leishmania) promastigote culture (final cell concentration 200,000 cells/ml) as a single addition in 96 well microtiter plates in complete DMEM media. The cultures were incubated for 24 h at 26°C, and the cell density was determined by the addition of the vital dye AlamarBlue® and the reduction of this dye was monitored over a period of an additional 24 h. After 48 hours, the cultures were examined microscopically to provide additional verification of the AlamarBlue® results.
[0173] It was also observed that treatment with several doses of inhibitory peptides (for example, three doses administered every 4 hours) had a greater effect on reduction of parasite viability.
[0174] The novel modulatory peptides disclosed herein were found to be effective against both the old and new world species. However, it is also understood that the treatment / modulatory peptide can be tailored to a particular Leishmania species.
[0102] Thus, the rationally designed peptides described herein exhibited anti-parasitic effects, inducing parasite cell death, reducing parasite viability, protecting macrophages from parasite infection and reducing the number of macrophage infected by the parasite, while having no toxic effect on host or control cells. Furthermore, the requirement for the parasite to develop two or more mutations to avoid susceptibility to a drug agent also dramatically reduces chance of parasitic resistance. These LACK / TRACK inhibitor peptides were also found to be effective against T. Cruzi and Chagas disease.
Therefore, in addition to their use as novel tools to study parasite function and viability, these peptide inhibitors are useful lead for the development of novel treatment of diseases, such as Cutaneous Leishmaniasis, Mucosal Leishmaniasis, Visceral
Leishmaniasis and Chagas disease. Thus, novel and effective approaches to rational
drug design has resulted in the identification of lead compounds useful for the treatment, amelioration and prevention of infectious diseases.
EXAMPLE 3
IDENTIFICATION OF NOVEL LACK- OR TRACK- BINDING PROTEINS
[0175] Leishmaniasis and trypanosomiasis are diseases caused by related parasites that affect millions of people mainly in the poorest countries and rural areas. Very few new drugs are currently under development and in general the current treatment relies on old, often toxic and ineffective drugs.
Identifying proteins that bind to LACK- and TRACK- derived peptides
[0176] To detect proteins that bound to the modulatory peptides, rationally designed peptides were coupled to a HiTrap™ NHS-activated HP column (GE Healthcare), and the column was washed. Next, the T. cruzi parasite lysates were passed three times through the resin alone to eliminate non-specific interactions and then to peptide- conjugated columns. Columns were washed and bound peptides / proteins were eluted from the column with glycine. Bound proteins were then concentrated, trypsin digested and identified by mass spectrometry. Below is a list of LACK- / TRACK- binding proteins which did not bind to the resin alone. It is noted that a modulatory peptide described herein bound elF1A, previously described to be a TRACK-binding protein. This is of interest because TRACK has been shown to be involved in protein translation in Trypanosoma brucei and RNAi for TRACK in T brucei inhibited cytokinesis, perhaps by inhibiting the translation of a protein that aids cytokinesis.
[0177] Proteins identified
gi| 162030 calmodulin A [Trypanosoma brucei]
gi|350663 calmodulin
gi|349838 heat shock protein [Trypanosoma cruzi]
gi|704459 elongation factor 1 alpha [Trypanosoma cruzi]
gi| 10626 Heat shock protein 70 [Trypanosoma cruzi]
gi|763030 Glucose-regulated protein 78 [Trypanosoma cruzi]
EXAMPLE 4
EFFECTS OF INHIBITORY PEPTIDES IN MICE IN VIVO
Peptide treatment of mice
[0178] A small in vivo study was performed in mice. Animal care and husbandry procedures were in accordance with established institutional and National Institutes of Health guidelines. The results of this study are shown in Figures 4-9 and a summary is
presented in Figure 10. Overall, modulatory peptide 341 was observed to reduce parasitemia in mice by 90% in this in vivo model compared to control (where the peak of the parasitemia was at day 9). Peptides 341 (cyclic) and 92 (linear) were found to increase survival in mice compared to the control.
Testing for toxicity of the peptides towards the host
[0179] The peptides used in these studies were also found to be non-toxic, and were observed not to have an effect on non-infected macrophages or other cultured cells. Next, we determined whether the cytotoxicity of the peptides is specific for the parasite. None of the peptides showed any toxicity in mammalian macrophage cells (data not shown) or to LLC-MK2 cells.
[0180] In the context of infected mammals it is important to have a therapeutic strategy that will also be effective towards the intracellular amastigote forms of T. cruzi. Peptides LLM-1.1 and LLM-2.2 were thus tested for their effect on infected macrophages. Peptides were not as effective towards amastigote forms as they were towards trypomastigote forms, addition of peptides 4X slightly increased their activity. One of the reasons for the low activity of the peptides towards amastigote forms is believed to be due to the fact that these peptides may be degraded by proteases; therefore, SAR studies were performed and cyclic peptides made to increase peptide stability. Peptide XXE was more effective towards promastigote forms of Leishmania donovani and Trypanosoma Cruzi and a single dose was able to decrease amastigote infection.
[0181] Thus, the technology of inhibiting key protein-protein interactions using peptides delivered chronically or acutely was shown to be successful in preventing myocardial infarction in various models including, in vitro, in culture and numerous in vivo animal models of different human diseases as well as in human clinical trials. Others have successfully shown that peptides can be delivered to Leishmania parasites and macrophages (Corradin, 2002 #2747). It can be difficult to chronically treat populations in endemic regions. Therefore, in animal studies, it is also important to test the effectiveness of the peptides when administered as an ointment, which is very useful for the treatment of Cutaneous Leishmaniasis. Moreover, as in vitro and in vivo animal models of the peptides are proven to be effective, modulatory peptides will serve as leads to the development of stable drugs that can be more feasibly administered to the population in an endemic region.
[0182] Finally, the probability for the development of drug resistance and desensitization to such peptides is lower for the modulatory peptides described herein. Drug resistance develops by mutations that alter drug binding-sites without affecting the biological function of the drug-target (Hastings, 2001 ). To acquire resistance to these
novel inhibitors, mutations that prevent their binding to LACK/TRACK without affecting the binding of the signaling molecule are unlikely to occur (Hastings, 2001 ). Moreover, therapy with the peptides can be combined with immunotherapy and available chemotherapy, and thus may reduce toxic effects related to the standard medication, since lower amounts of toxic chemotherapeutics could be used.
[0183] The presently described rationally designed peptides derived from unique sequences can induce selective cytotoxic effect on promastigotes without affecting host cell viability or altering RACK-dependent functions in mammalian cells.
[0184] The inhibitory peptides disclosed herein have several advantages:
1. They are the first selective regulators of protein-protein interactions found to reduce parasite viability and macrophage infection by the parasite.
2. Treatment with these peptides induced promastigotes' cell death, and reduced macrophage infection.
3. Treatment with these peptides protected macrophage cells from parasite infection, reducing cell death. The peptides exerted no effects on macrophage cells function or macrophage cell viability in healthy cells. These data indicated that the peptide inhibitors had anti-parasitic effects without affecting the host cells.
4. These peptides are linked to Tat47-57, which enables penetration of the peptides into cells and across both macrophages and parasites. Therefore, these peptides are believed to be useful for the treatment of these neglected tropical diseases.
5. Treatment of humans with peptide similar in molecular weight and charge to these ones was found to be safe and efficacious.
6. The peptides designed to inhibit Leishmania's protein-protein interactions were also effective against T. Cruzi, suggesting that a single chemotherapeutic treatment can be used for these two different parasites.
7. The peptides can be used in a screen to identify small molecule inhibitors.
[0185] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.
Claims
1. A modulatory peptide that selectively modulates the binding of a LACK or TRACK protein to its cognate binding protein, comprising a contiguous sequence of at least 5 amino acid residues, wherein said contiguous sequence is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.
2. The modulatory peptide of claim 1 , wherein the modulatory peptide consists of 5- 20 amino acid residues.
3. The modulatory peptide of claim 1 , wherein the modulatory peptide is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.
4. The modulatory peptide of claim 2, wherein the modulatory peptide is at least 95% identical to a portion of calmodulin A (SEQ ID NO: 15).
5. The modulatory peptide of claim 2, wherein the modulatory peptide is at least 95% identical to a portion of calmodulin (SEQ ID NO: 16).
6. The modulatory peptide of claim 2, wherein the modulatory peptide is at least 95% identical to a portion of heat shock protein (SEQ ID NO: 17).
7. The modulatory peptide of claim 2, wherein the modulatory peptide is at least 95% identical to a portion of elongation factor 1 alpha (SEQ ID NO: 18).
8. The modulatory peptide of claim 2, wherein the modulatory peptide is at least 95% identical to a portion of heat shock protein 70 (SEQ ID NO: 19).
9. The modulatory peptide of claim 2, wherein the modulatory peptide is at least 95% identical to a portion of glucose-regulated protein 78 (SEQ ID NO: 20).
10. A modulatory composition comprising the peptide of any one of claims 1-9.
1 1. The composition of claim 10, further comprising a pharmaceutically acceptable excipient.
12. A method for reducing parasite infectivity comprising administering to a subject in need thereof a modulatory peptide of any one of claims 1 -9.
13. The method of claim 12, wherein the subject is suffering from subject is suffering from a disease selected from the group consisting of cutaneous leishmaniasis, mucosal leishmaniasis, visceral leishmaniasis, Chagas disease and trypanosomiasis.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261693777P | 2012-08-28 | 2012-08-28 | |
US61/693,777 | 2012-08-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2014036198A1 true WO2014036198A1 (en) | 2014-03-06 |
Family
ID=50184332
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2013/057175 WO2014036198A1 (en) | 2012-08-28 | 2013-08-28 | Compositions and methods for specific inhibition of leishmania receptor for activated c-kinase (lack) |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2014036198A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2021514940A (en) * | 2018-02-27 | 2021-06-17 | リポトゥルー,エセ.エレ. | Peptides and compositions for use in cosmetics and medicine |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010129267A2 (en) * | 2009-04-27 | 2010-11-11 | University Of Georgia Research Foundation, Inc. | Anti-trypanosomal peptides and uses thereof |
-
2013
- 2013-08-28 WO PCT/US2013/057175 patent/WO2014036198A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010129267A2 (en) * | 2009-04-27 | 2010-11-11 | University Of Georgia Research Foundation, Inc. | Anti-trypanosomal peptides and uses thereof |
Non-Patent Citations (12)
Title |
---|
BENAIM GUSTAVO ET AL.: "Comparative phosphorylation of calmodulin from trypanosomatids and bovine brain by calmodulin-binding protein kinases.", COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY, vol. 120, 1998, pages 57 - 65 * |
DATABASE GENBANK 16 July 2001 (2001-07-16), accession no. AK51528.1 * |
DATABASE GENBANK 17 August 1994 (1994-08-17), accession no. AA30201 * |
DATABASE GENBANK 18 April 2005 (2005-04-18), accession no. AA47952 * |
DATABASE GENBANK 26 April 1993 (1993-04-26), accession no. AA30174.1 * |
DATABASE NCBI 20 May 2009 (2009-05-20) * |
DATABASE PRF 19 June 1996 (1996-06-19), accession no. 50663 * |
EICHHOLTZ THOMAS ET AL.: "A Myristoylated Pseudosubstrate Peptide, a Novel Protein Kinase C Inhibitor.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 3, 25 January 1993 (1993-01-25), pages 1982 - 1986 * |
KIANG JULIANN G. ET AL.: "Heat Shock Protein 70 kDa: Molecular Biology, Biochemistry, and Physiology.", PHARMACOL. THER., vol. 80, no. 2, 1998, pages 183 - 201 * |
MELBY PETER C. ET AL.: "Leishmania donovani p36 (LACK) DNA Vaccine Is Highly Immunogenic but Not Protective against Experimental Visceral Leishmaniasis.", INFECTION AND IMMUNITY, vol. 69, no. 8, 2001, pages 4719 - 4725 * |
VENEMA RICHARD C. ET AL.: "Phosphorylation of Elongation Factor 1 (EF-1) and Valyl-tRNA Synthetase by Protein Kinase C and Stimulation of EF-1 Activity.", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 19, 5 July 1991 (1991-07-05), pages 12574 - 12580 * |
WEISS GREGORY A. ET AL.: "Rapid mapping of protein functional epitopes by combinatotial alanine scanning", PNAS, vol. 97, no. 16, 2000, pages 8950 - 8954 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2021514940A (en) * | 2018-02-27 | 2021-06-17 | リポトゥルー,エセ.エレ. | Peptides and compositions for use in cosmetics and medicine |
JP7296642B2 (en) | 2018-02-27 | 2023-06-23 | リポトゥルー,エセ.エレ. | Peptides and compositions for cosmetic and pharmaceutical use |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20140329750A1 (en) | Growth Hormones with Prolonged In-Vivo Efficacy | |
CN104797603A (en) | Peptide directed protein knockdown | |
EP2368898B1 (en) | Compositions and methods for counteracting effects of reactive oxygen species and free radicals | |
EP3829621A1 (en) | Engineered hemichannels, engineered vesicles, and uses thereof | |
CN110944677A (en) | Topical compositions and uses | |
EP3684783B1 (en) | Gene expression inhibitors | |
CN110248953A (en) | Novel stapler peptide and application thereof | |
BRPI0613849A2 (en) | polypeptide and pharmaceutically acceptable salts or esters, pharmaceutical composition and use of the polypeptide | |
Qvit et al. | Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors | |
US8940868B2 (en) | Elastin based growth factor delivery platform for wound healing and regeneration | |
US9567369B2 (en) | Method of treating metastatic cancer | |
WO2014036198A1 (en) | Compositions and methods for specific inhibition of leishmania receptor for activated c-kinase (lack) | |
EP3210995A1 (en) | Hemagglutinin-binding peptide | |
EP2004680B1 (en) | N-terminal vdac variants and uses thereof | |
JP2010239971A (en) | Promotion of peroxisomal catalase function in cell | |
KR20120001964A (en) | Peptides for treatment and prevention of allergic diseases | |
CA2791058A1 (en) | Methods for inhibiting necrosis | |
US10131691B2 (en) | Compositions and methods for specific regulation of pyruvate dehydrogenase kinase | |
CN102675424B (en) | Polypeptide drug for resisting Hepatitis B virus X protein | |
US9815867B2 (en) | Peptide for inhibiting vascular endothelial growth factor receptor | |
US20240228543A1 (en) | Cyclic peptide inhibitors of usp22 | |
KR101942755B1 (en) | Method for producing melittin-like recombinant protein and composition for skin containing them | |
AU2021480402A1 (en) | Sestrin-mapk complex inhibitors | |
US20140155331A1 (en) | Novel high affinity bivalent helically constrained peptide against cancer | |
EP4313110A1 (en) | K-ras inhibitor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13834146 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 13834146 Country of ref document: EP Kind code of ref document: A1 |