WO2006084471A2 - Methode de conception de medicament rationnelle fondee sur la capacite de liaison au facteur d'allongement tu - Google Patents

Methode de conception de medicament rationnelle fondee sur la capacite de liaison au facteur d'allongement tu Download PDF

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WO2006084471A2
WO2006084471A2 PCT/DK2006/000077 DK2006000077W WO2006084471A2 WO 2006084471 A2 WO2006084471 A2 WO 2006084471A2 DK 2006000077 W DK2006000077 W DK 2006000077W WO 2006084471 A2 WO2006084471 A2 WO 2006084471A2
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WO2006084471A3 (fr
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Poul Nissen
Andrea Parmeggiani
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Aarhus Universitet
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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/50—Molecular design, e.g. of drugs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Definitions

  • a method of rational drug design based upon the surprising discovery that the structurally very different antibiotics enacyloxin Ha and kirromycin bind with Elongation Factor Tu (EF- Tu) in a similar manner allows for new chemical entities and new antibiotics to be developed.
  • the invention defines new antibiotics which are structural hybrids of enacyloxin Ha and kirromycin; the new antibiotics combining critical moieties of each of the parent molecules.
  • US 6,684,162 discloses the crystal structure of Acyl Carrier Protein Synthase (ACPS) complexed with Acyl Carrier Protein (ACP) and the solution structure of B. subtilis ACP. Further disclosed is a method for rational drug design based on the crystal shape and dimensions, wherein the active site is identified from the three-dimensional model of the structure and subsequent computer fitting is used to identify possible antibacterial agents.
  • ACPS Acyl Carrier Protein Synthase
  • ACP Acyl Carrier Protein
  • B. subtilis ACP B. subtilis ACP
  • WO 03/060100 discloses DnaG primase proteins complexed with manganese and sulfate ligands. Further disclosed is a method of rational drug design based on the atomic coordinates of the three-dimensional crystal structure of the complex, selecting potential antibacterial agents with computer modelling and subsequent testing of the activity.
  • EP 1 302 538 discloses the three-dimensional crystal structure of human sorbitol dehydrogenase (hSDH) complexed with a ligand. This structure again serves as a basis for a computer based method of identifying new ligands.
  • hSDH human sorbitol dehydrogenase
  • the present invention compares and contrasts the structural relationship of two key ligands to EF-Tu to their complexation and activity on the protein. This comparison is possible in the present case due to the unique and surprising discovery that the structurally different ligands bind EF-Tu in such a similar manner. Yet, at once, the differences in the binding pattern allow for contrasting of the ligands.
  • Elongation factor (EF-) Tu a ubiquitous, highly conserved, abundant protein, is an essential component of bacterial protein synthesis and one of the best studied GTPases. GTPases cycle between an active GTP-bound and an inactive GDP-bound state, that is controlled by the GDP/GTP exchange and GTP hydrolysis, two reactions regulated in EF-Tu by EF-Ts and the ribosome, respectively.
  • EF-Tu «GTP is the carrier of aa-tRNA to the mRNA-programmed ribosome, where codon-anticodon interaction triggers a fast ⁇ - phosphate hydrolysis of GTP.
  • codon-anticodon interaction triggers a fast ⁇ - phosphate hydrolysis of GTP.
  • EF-Tu is thus an important target for inhibitors of protein synthesis.
  • Thermus thermophilus (Tt) EF-Tu»GDP in complex with methylkirromycin confirmed that the drug binds in the domain-1,3 interface, inducing a unique, compact EF-Tu* GTP- 1 ike conformation (Vogeley et al., 2002).
  • This invention is a result, at least in part, of the surprising discovery of the similar binding of enacyloxin Ha and kirromycin to of EF-Tu.
  • the present inventors have investigated a method of rational drug design comprising combination of the "head and body of enacyloxin Ha” and "hydrophobic tail of kirromycin", compounds resulting from the design, and a library comprising a plurality of said hybrid compounds. Furthermore, an assay for screening said library and computer based method of generating said compounds have been investigated.
  • a first aspect of the invention is a method of rational drug design comprising combining structural features of enacyloxin Ha and kirromycin to arrive at a compound of formula Ha,
  • Z comprises a moiety capable of making a dipole-dipole interaction with Lys313, another moiety capable of forming a hydrogen bond with Tyrl60, and Z should provide the required distance between these two moieties when taking hydrogen bond lengths and the distance between Lys313 and Tyrl60 into account;
  • LM 2 are independently absent or selected from the group consisting of O and NH;
  • L 3 is O or S;
  • R 4 , R 5 are independently selected from H, AIk, Cl, Br, and I;
  • K comprises hydrogen bonding moiety(ies) capable of binding to Argl23 and Glnl24;
  • n is sufficiently large to provide the necessary distance between the binding moieties in Z and K when taking hydrogen bond lengths and the distance between Lys313 and Tyrl ⁇ O on one side and Argl23 and Glnl24 on the other side into account;
  • V is selected from CH 2 , NH, O, C(O), (CH 2 ) q -C(O); O-C(O), O-C(O)-O(CH 2 ) q -C(O)-O,
  • R 8 is selected from AIk, OH, OAIk, NH 2 , F, Cl, Br, and I; m is 1 or 2; q is an integer selected from 1, 2, 3, 4, 5, 6, and 7; I is an integer from 3 to 7; and
  • X is hydrophobic and fits into the hydrophobic pocket delimited by Ile92, Vall25, and
  • a second aspect of the invention is a method for rational drug design by evaluating the potential of a chemical entity to associate with: a) a molecule or molecular complex comprising a binding pocket defined by structure coordinates of EF-Tu domain 1 and 3 amino acids Arg 123, Glnl24, Vall25, Tyri60, Arg333, Arg373, Phe374, and Ala375 according to table III; or b) a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Angstroms comprising the steps of:
  • a third aspect of the invention is a compound of the formula I:
  • Z is absent or an optionally substituted aromatic or non-aromatic, cyclic or heterocyclic ring structure with 4 - 10 atoms in the ring(s) selected from C, O, N, and S;
  • L is a linker which is absent or selected from the group consisting of -C(O)-, -
  • Fat comprises an optionally substituted straight aliphatic chain with 2 - 20 carbon atoms, optionally having 1 to 4 carbon atoms replaced by nitrogen, oxygen, or sulphur, which has fully conjugated double bonds, is mono- or polyunsaturated, or saturated;
  • Y comprises a 5-, 6-, or 7-membered cyclic or heterocyclic ring, the heteroatoms being selected from O, N, and S, substituted with 1 to 10 substituents selected from the group consisting of AIk, OH, NH 2 , F, Cl, Br, and I, preferred substituents being methyl and OH;
  • X is an optionally substituted straight aliphatic chain with 2 to 10 carbon atoms, such as 2 to 8, optionally having fully conjugated double bonds and one or more carbon atoms replaced by O, N, or S.
  • the compound of formula I is a compound of formula Ha:
  • L 1 , L 2 are independently absent or selected from the group consisting of O and NH; is O or S;
  • R 4 R b are independently selected from H, AIk, Cl, Br, and I;
  • K comprises hydrogen bonding moiety(ies) capable of binding to Argl23 and Glnl24; n is sufficiently large to provide the necessary distance between the binding moieties in Z and K when taking hydrogen bond lengths and the distance between Lys313 and Tyrl ⁇ O on one side and Argl23 and Glnl24 on the other side into account;
  • V is selected from CH 2 , NH, O, C(O), (CH 2 ) q -C(O); O-C(O), O-C(O)-O(CH 2 ) q -C(O)-O,
  • R 8 is selected from AIk, OH, OAIk, NH 2 , F, Cl, Br, and I; m is 1 or 2; q is an integer selected from 1, 2, 3, 4, 5, 6, and 7; is an integer from 3 to 7; and X is hydrophobic and fits into the hydrophobic pocket delimited by Ile92, Vall25, and
  • a further aspect is a library comprising a plurality of species of conjugate compounds defined supra.
  • an important aspect of the invention relates to the use of any one of the compounds from said library of conjugate compounds as ligands for EF-Tu or for the preparation of antibiotics.
  • aspects of the invention include a method of inhibiting EF-Tu activity comprising administering a compound of formula I, II, III, IV, or V as defined supra and a method of providing antibacterial effect in or on a human or animal comprising administering a compound of formula I, II, III, IV, or V as defined supra.
  • Figure 1 shows a superposition of the structures of Enacyloxin Ha and Kirromycin in complex with EF-Tu.
  • the middle "body” parts are roughly in the same plane and together they appear to be forming a ring structure.
  • Kirromycin is surrounded ( ⁇ 3.8 A) by 19 amino acids (8 from domain 1 and 11 from domain 3), of which 4 from domain 1 and 4 from domain 3 are common to enacyloxin Ha, and makes five hydrogen bonds with EF-Tu, of which one is shared by two atomic groups of the same amino acid (Argl23(124) N ⁇ and N ⁇ l).
  • the three hydrogen-bonding residues, Argl23(I24), Glnl24(125J and Tyrl60(161) are in common with enacyloxin Ha.
  • Tyrl60(I6_?) hydroxy is part of a three-centered hydrogen bond with Argll6(117) and the pyridone ring OH(4) of kirromycin, as with the hexane-carboxy group of enacyloxin Ha.
  • Kirromycin is practically wrapped around the aliphatic chain of Glnl24(125j with several contacts, whereas enacyloxin Ha steers around this residue.
  • the "heads" of enacyloxin IIa and kirromycin point in opposite directions at right angles to the body of the antibiotics
  • Glnl24 in EF-Tu(Ec) and Glnl25 in EF-Tu(Ec) occupy substantially the same position in the enacyloxin IIa and kirromycin complexes respectively.
  • the distance is defined in terms of the coordinates given for the atoms of the amino acid residues in the complexed EF-Tu in Table III.
  • binding distances the distances are given for the atoms in the amino acid residues of EF-Tu that do the actual binding.
  • a unique feature of the enacyloxin Ha binding is the presence of a salt bridge linking the carboxyl group of the hexane ring with Lys313 N ⁇ , but the most important difference from kirromycin concerns its short hydrophobic tail that borders but does not enter the near hydrophobic pocket lacking ordered water molecules, whereas the longer hydrophobic tail of kirromycin fits this pocket. This divergence is a major reason for the lower binding affinity of enacyloxin Ha, that is evident by comparing the ability of the two antibiotics to restore the EF-Tu-dependent poly(Phe) synthesis after gel filtration.
  • One aspect of the invention is a method of rational drug design comprising combining structural features of enacyloxin Ha and kirromycin to arrive at a compound of formula Ha,
  • Z comprises a moiety capable of making a dipole-dipole interaction with Lys313, another moiety capable of forming a hydrogen bond with Tyrl ⁇ O, and Z should provide the required distance between these two moieties when taking hydrogen bond lengths and the distance between Lys313 and TyrlGO into account;
  • L 1 , L 2 are independently absent or selected from the group consisting of O and NH;
  • L 3 is O or S;
  • R 4 , R 5 are independently selected from H, AIk, Cl, Br, and I; 6 000077
  • K comprises hydrogen bonding moiety(ies) capable of binding to Argl23 and Glnl24; n is sufficiently large to provide the necessary distance between the binding moieties in Z and K when taking hydrogen bond lengths and the distance between Lys313 and Tyrl60 on one side and Argl23 and Glnl24 on the other side into account; V is selected from CH 2 , NH, O, C(O), (CH 2 ) q -C(O); O-C(O), O-C(O)-O(CH 2 ) q -C(O)-O,
  • R 8 is selected from AIk, OH, OAIk, NH 2 , F, Cl, Br, and I; m is 1 or 2; q is an integer selected from 1, 2, 3, 4, 5, 6, and 7;
  • I is an integer from 3 to 7;
  • X is hydrophobic and fits into the hydrophobic pocket delimited by Ile92, Vall25, and
  • this method is a computer based method of generating compounds for rational drug design having a prescribed set of structural features, the method comprising the steps of:
  • the invention relates to a method for rational drug design by evaluating the potential of a chemical entity to associate with: a) a molecule or molecular complex comprising a binding pocket defined by structure coordinates of EF-Tu domain 1 and 3 amino acids Arg 123, Glnl24, Vall25, Tyrl ⁇ O, Arg333, Arg373, Phe374, and Ala375; or b) a homologue of said molecule or molecular complex, wherein said homologue comprises a binding pocket that has a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Angstroms comprising the steps of:
  • said chemical entity can associate with said binding pocket such that the binding distances with amino acids Argl23, Glnl24, and Tyrl60 are less than 3.75 Angstroms, such as 2.0 to 3.5 Angstroms, and binding distances with amino acids Vali25, Arg333, Arg373, Phe374, and Ala375 are 2 to 3.8 Angstroms, preferably 3.5 to 3.8 Angstroms.
  • an important aspect of the invention relates to the use of any one of the compounds from said library of conjugate compounds as ligands for EF-Tu or for the preparation of antibiotics.
  • aspects of the invention include a method of inhibiting EF-Tu activity comprising administering a compound of formula I, II, III, IV, or V as defined supra and a method of providing antibacterial effect in or on a human or animal comprising administering a compound of formula I, II, III, IV, or V as defined supra.
  • a still further aspect is a high throughput assay comprising testing said library for activity towards EF-Tu.
  • One particularly interesting embodiment is the computer-based method for rational drug design defined supra comprising said assay.
  • AIk is throughout this application intended to mean C 1-4 alkyl, such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, or tert-butyl.
  • substituents such as 1 to 5 substituents, preferably 1 to 3 substituents, most preferably 1 to 2 substituents, which may be selected from hydroxy, Ci_ e -alkyl, C 1-5 -alkoxy, C 2 - 6 -alkenyloxy, oxo, carboxy, C 1-6 -alkoxycarbonyl, C],- 6 -alkylcarbonyl, formyl, aryl, aryloxy, arylamino, aryloxycarbonyl, arylcarbonyl, heteroaryl, heteroarylamino, amino, mono- and di(C 1 .
  • substituents such as 1 to 5 substituents, preferably 1 to 3 substituents, most preferably 1 to 2 substituents, which may be selected from hydroxy, Ci_ e -alkyl, C 1-5 -alkoxy, C 2 - 6 -alkenyloxy, oxo, carboxy, C 1-6 -alkoxycarbonyl, C],
  • halogen includes fluorine, chlorine, bromine and iodine.
  • binding pocket refers to a region of a molecule or molecular complex, that, as a result of its shape, favourably associates with another chemical entity or compound.
  • binding pockets many drugs exert their biological effects through association with the binding pockets of receptors and enzymes. Such associations may occur with all or any parts of the binding pockets.
  • association refers to a condition of proximity between chemical entities or compounds, or portions thereof.
  • the association may be non-covalent — wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions — or it may be covalent.
  • Another aspect of the invention is a compound of the formula I:
  • Z is absent or an optionally substituted aromatic or non-aromatic, cyclic or heterocyclic ring structure with 4 - 10 atoms in the ring(s) selected from C,
  • L is a linker which is absent or selected from the group consisting of -C(O)-, -
  • Fat comprises an optionally substituted straight aliphatic chain with 2 - 20 carbon atoms, optionally having 1 to 4 carbon atoms replaced by nitrogen, oxygen, or sulphur, which has fully conjugated double bonds, is mono- or polyunsaturated, or saturated;
  • Y comprises a 5-, 6-, or 7-membered cyclic or heterocyclic ring, the heteroatoms being selected from O, N, and S, substituted with 1 to 10 substituents selected from the group consisting of AIk, OH, NH 2 , F, Cl, Br, and I, preferred substituents being methyl and OH;
  • X is an optionally substituted straight aliphatic chain with 2 to 10 carbon atoms, such as 2 to 8, optionally having fully conjugated double bonds and one or more carbon atoms replaced by O, N, or S.
  • the compounds of the present invention need not merely be a simple hybrid of Kirromycin and Enacyloxin Ha wherein a part of one molecule is pieced together with the opposite part of the other molecule. It is contemplated that it may also be a superposition of the two central parts of Kirromycin and Enacyloxin Ha respectively, i.e. a cyclic structure.
  • X while being an optionally substituted straight aliphatic chain with 2 to 10 carbon atoms, optionally having fully conjugated double bonds and one or more carbon atoms replaced by O, N, may form part of ring system, optionally fused with Y.
  • the compound of formula I may have the formula II:
  • Z is a group of formula:
  • W is selected from the group consisting of CH 2 , O, NH, CH 2 -CH 2 , CH 2 -O, CH 2 -NH, CH 2 -CH 2 -CH 2 , CH 2 -CH 2 -O, and CH 2 -CH 2 -NH;
  • R 1 , R 2 , R 3 are independently selected from the group consisting of H, AIk, AIk-C(O), AIk-C(O)-O, AIk-O-C(O), NH 2 , NHAIk, N(AIk) 2 , NH 2 -C(O), NHAIk-C(O), N(AIk) 2 -C(O), OH, OAIk, F, Cl, Br, and i;
  • LM 2 are independently absent or selected from the group consisting of O and NH; is O or S; is an integer from 4 to 8;
  • R 4 , R 5 are independently selected from H, AIk, Cl, Br, and I;
  • K comprises C 1-6 alkylidene or a mono-, fused bi-, tri-, or tetracyclic structure with 12 to 20 ring members substituted 1 to 6 times, such as 2, 3, or 4 times with AIk, OH,
  • OAIk, NH 2 , or F with the proviso that at least one, such as at least two, of the substituents is/are selected from OH, OAIk, NH 2 , and F, preferably OH; wherein one or more of the rings of said ring structure may contain one or more double bonds and/or contain a group -0-, -NH-, -S-, -C(O)-O-, or -O-C(O)-;
  • V is selected from CH 2 , NH, O, C(O), (CH 2 ) q -C(O); O-C(O), O-C(O)-O(CH 2 ) q -C(O)-O,
  • R 8 is selected from AIk, OH, OAIk, NH 2 , F, Cl, Br, and I; m is 1 or 2; q is an integer selected from 1, 2, 3, 4, 5, 6, and 7;
  • I is an integer from 3 to 7; and X is an optionally substituted straight aliphatic chain with 2 to 8 carbon atoms, such as 4, 5, or 6, optionally with fully conjugated double bonds.
  • X while being an optionally substituted straight aliphatic chain with 2 to 10 carbon atoms, optionally having fully conjugated double bonds and one or more carbon atoms replaced by O, N, may form part of ring system, optionally fused with Y.
  • the compound of formula I may have the formula Ha:
  • Z comprises a moiety capable of making a dipole-dipole interaction with Lys313, another moiety capable of forming a hydrogen bond with Tyrl ⁇ O, and Z should provide the required distance between these two moieties when taking hydrogen bond lengths and the distance between Lys313 and Tyrl60 into account;
  • L 1 , L 2 are independently absent or selected from the group consisting of O and NH;
  • L 3 is O or S
  • R 4 , R 5 are independently selected from H, AIk, Cl, Br, and I;
  • K comprises hydrogen bonding moiety(ies) capable of binding to Argl23 and Glnl24; n is sufficiently large to provide the necessary distance between the binding moieties in Z and K when taking hydrogen bond lengths and the distance between Lys313 and Tyrl60 on one side and Argl23 and GIn 124 on the other side into account;
  • V is selected from CH 2 , NH, O, C(O), (CH 2 ) q -C(O); O-C(O), O-C(O)-O(CH 2 ) q -C(O)-O,
  • R 8 is selected from AIk, OH, OAIk, IMH 2 , F, Cl, Br, and I; m is 1 or 2; q is an integer selected from 1, 2, 3, 4, 5, 6, and 7;
  • I is an integer from 3 to 7;
  • X is hydrophobic and fits into the hydrophobic pocket delimited by Ile92, VaI 125, and Ala375.
  • the compounds of formula I, II or Ha may have the formula III:
  • W is selected from the group consisting of CH 2 , O, and NH;
  • R' are independently selected from the group consisting of H, AIk, AIk-C(O), AIk-C(O)-
  • L 1 is absent or selected from the group consisting of O and NH; n is an integer from 4 to 8;
  • V is selected from CH 2 , NH, O, C(O), (CH 2 ) q -C(O); O-C(O), O-C(O)-O(CH 2 ) q -C(O)-O,
  • R 8 is selected from methyl, ethyl, OH, methoxy, and ethoxy; I is an integer from 3 to 7; and q is an integer selected from 1, 2, 3, 4, 5, 6, and 7; j is an integer from 1 to 4.
  • the compounds of formula I, II or III may have the formula IV:
  • R 1 , R 2 are independently selected from the group consisting of H, AIk, AIk-C(O), AIk-C(O)-
  • L 1 is absent or selected from the group consisting of O and NH; n is 5, 6, or 7; R 4 are independently selected from H and methyl, with the proviso that at least one R 4 is methyl; R 5 are independently selected from H and Cl, with the proviso that at least one R 5 is
  • R 7 is not H
  • V is selected from CH 2 , NH, O, C(O), (CH 2 ) q -C(O); O-C(O), O-C(O)-O(CH 2 ) q -C(O)-O,
  • R 8 is selected from methyl, ethyl, OH, methoxy, and ethoxy; q is an integer selected from 1, 2, 3, 4, 5, 6, and 7;
  • I is 4, 5, or 6; and i is 2 or 3.
  • a further aspect is a library comprising a plurality of species of conjugate compounds defined supra.
  • EF-Tu-GDP After Escherichia coli [Ec) cell extraction by French press, EF-Tu-GDP was purified by centrifugation at 30,00Og (S30) and 10O 7 OOOg (SlOO), FPLC on Source-Q, filtration on Superdex 75 and HPLC on monoQ. Purified EF-Tu yielded a single band on SDS-PAGE and
  • EF-Tu T ⁇ erm us aquaticus: Ta was overproduced in E. coli and purified as described (Kristensen et ah,
  • EF-Tu-GDPNP was obtained from EF-Tu-GDP in 20 mM Tris-HCI, pH 8.0, 200 mM
  • the solution for crystallization contained 4-4.5 mg/ml EF-Tu in 20 mM Tris-HCI buffer (pH 7.6), 20 mM NaCI, a 2-fold molar excess of GDPNP, 5-10 mM MgCl 2 , 2 mM DTT, 1 % glycerol and 0.5 mM NaN 3 .
  • the antibiotic added as the last component in a 1.2 molar ratio to EF-Tu, caused a slight precipitate that was discarded by centrifugation.
  • a molar ratio enacyloxin Ha EF-Tu close to 1 is crucial, since higher ratios caused aggregation and precipitation of the protein. No hydrolysis of the crystalline EF-Tu was observed on SDS-PAGE.
  • the complex EF-Tu(7a)»GDPNP»enacyloxin IIa»Phe-tRNA phe (S. cerevisiae) was purified and crystallized essentially as described for the quaternary complex with kirromycin (Kristensen et a/.).
  • An ammonium sulfate precipitated aliquot was redissolved to 10 mg/ml in a crystallization buffer containing 1.25 M (NH 4 ) 2 SO 4 , 10 mM MgCI 2 , 20 mM Tris-HCI (pH 7.2 at 20 0 C), 1 mM GDPNP, 0.5 mM DTT, and 50 ⁇ M enacyloxin Ha.
  • Hanging drops (3-8 ⁇ l) were equilibrated at 4 0 C against reservoir solutions containing 1 ml of 1.8 M ammonium sulfate (pH 6.4 -7.0).
  • Model-derived phases were calculated at 30 - 2.3 A resolution using ⁇ A weighting and were subjected to refinement by density modification using two-fold averaging, solvent flattening and histogram matching in DM (CCP4, 1994) and using phase extension from 3.5 to 2.3 A resolution over 100 cycles.
  • the resulting DM map showed clear electron density for most parts of EF-Tu»GDPNP and enacyloxin Ha.
  • the EF-Tu»GDPNP»enacyloxin IIa»Phe-tRNA Phe structure contained three molecules in the asymmetric unit related by three-fold NCS as observed also for the Phe-tRNA phe »EF-Tu « GDP»kirromycin complex.
  • Atomic coordinates for the EF-Tu»GDPNP «enacyloxin IIa»Phe- tRNA phe structure are given in Table III.
  • Methylkirromycin (N: 19) Ile92(93), Glull7(J18), Leul20(_!22), Argl23(i24), Glnl24(125), Vall25(126), Tyrl60(161), Glul62Tyr309(32_!), Asp324(G/u326), Glu325(327), Arg318(330), His319(33I), Arg333 ⁇ 345), Arg373(385), Phe374(386), Ala375(3S7),
  • Enacyloxin Ha (no: 4) Leul20 O-Argl23 N; Arg 123 N ⁇ 2-Glu315 O; Ala 375 O-Tyr331 IM; Ala375 O-Tyr331 N
  • Model building was performed in O.
  • Four possible models for enacyloxin Ha were derived by energy-minimization and they combined R or S configurations on the positions C13 and C14.
  • the C13-R, C14-S configuration revealed the best fit to the DM map of the EF- Tu:GDPNP «enacyloxin IIa complex, and this assignment was adapted.
  • Model refinement was carried out in CNS.
  • the initial refinement of the EF- Tu»GDPNP»Enacyloxin Ha model derived from the DM map was performed at 5 - 2.3 A resolution with strict NCS and without bulk solvent correction to avoid bias from a partial model.
  • a set of 1203 randomly selected reflection were excluded throughout for calculation of the free R-value.
  • Subsequent rounds of refinements included all data and a bulk solvent correction together with NCS restraints.
  • the ⁇ A weighted 2Fo-Fc and Fo-Fc electron density maps were used in later rounds of model building.
  • the final model comprises amino acid residues 9-40 and 55-393 for chain A and residues 6-40 and 57-393 for chain B as well as complete models of GDPNP»Mg 2+ and enacyloxin Ha for both complexes.
  • a total of 198 water molecules identified at appropriate positions for interactions with the macromolecular components were included in the final model, and the final R-free of the model was 27.2% at 30 - 2.3 A resolution.
  • the Phe-tRNA Phe »EF-Tu «GDPNP»enacyloxin Ha complex was refined at 30 - 3.0 A resolution as a single protomer obeying strict three-fold NCS throughout.
  • the final model comprises a complete Phe-tRNA molecule, EF-Tu(Ta) residues 9-405, GDPNP»Mg 2+ and enacyloxin Ha.
  • the final R-free converged at 30.7%.
  • ATOM 281 C ILE A 60 21.401 63.583 66.420 1.00 78.42 A
  • ATOM 307 CA THR A 64 25.690 58.256 63.035 1.00 80.33 A
  • ATOM 402 NDl HIS A 75 25.188 67.036 77.011 1.00 33.33 A
  • ATOM 668 C MET A 112 48.214 66.230 54.803 1.00 50.54 A
  • ATOM 702 C ARG A 116 44.518 71.608 54.973 1.00 42.64 A ATOM 703 O ARG A 116 44.066 72.686 55.366 1.00 42.99 A
  • ATOM 761 O ARG A 123 38.079 80.903 58.030 1.00 38.38 A ATOM 762 N GLN A 124 38.264 79.288 56.497 1.00 33.44 A
  • ATOM 814 C ILE A 130 42.065 74.097 68.217 1.00 33.29 A
  • ATOM 822 C ILE A 131 43.143 70.976 68.906 1.00 29.65 A

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  • Bioinformatics & Computational Biology (AREA)
  • Theoretical Computer Science (AREA)
  • Evolutionary Biology (AREA)
  • Biophysics (AREA)
  • Medical Informatics (AREA)
  • Biotechnology (AREA)
  • Computing Systems (AREA)
  • Saccharide Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une méthode de conception de médicament rationnelle. Cette méthode est fondée sur la découverte surprenante selon laquelle les antibiotiques énacyloxine Ha et kirromycine, qui sont structurellement très différents, se lient au facteur d'allongement Tu (EF-Tu) de manière analogue. Ceci permet à de nouvelles entités chimiques et à de nouveaux antibiotiques d'être élaborés. L'invention concerne une méthode pour concevoir de nouveaux antibiotiques qui sont des hybrides structuraux de l'énacyloxine IIa et de la kirromycine. Ces antibiotiques combinent des fractions critiques de chaque molécule parent.
PCT/DK2006/000077 2005-02-09 2006-02-09 Methode de conception de medicament rationnelle fondee sur la capacite de liaison au facteur d'allongement tu WO2006084471A2 (fr)

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DKPA200500189 2005-02-09
DKPA200500189 2005-02-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021155459A1 (fr) * 2020-02-03 2021-08-12 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Procédés améliorés de conception rationnelle et de production de biomolécules

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Elongation Faktor-TU" [Online] 12 December 2005 (2005-12-12), , XP002409954 Retrieved from the Internet: URL:http://www.chem.umass.edu/~cmartin/Cou rses/BioStruct/2005/StudentPresentations/S hivKumarRedhu/W_EFTu_ShivKumar.pdf> [retrieved on 2006-11-27] the whole document *
GSCHWEND D A ET AL: "MOLECULAR DOCKING TOWARDS DRUG DISCOVERY" JOURNAL OF MOLECULAR RECOGNITION, HEYDEN & SON LTD., LONDON, GB, vol. 9, 1996, pages 175-186, XP000882526 ISSN: 0952-3499 *
HOGG T ET AL: "INHIBITORY MECHANISMS OF ANTIBIOTICS TARGETING ELONGATION FACTOR TU" CURRENT PROTEIN AND PEPTIDE SCIENCE, BENTHAM SCIENCE PULBISHERS, NL, vol. 3, no. 1, February 2002 (2002-02), pages 121-131, XP008024024 ISSN: 1389-2037 *
HONMA T: "Recent advances in de novo design strategy for practical lead identification" MEDICINAL RESEARCH REVIEWS, NEW YORK, NY, US, vol. 23, no. 5, September 2003 (2003-09), pages 606-632, XP002326365 ISSN: 0198-6325 *
PARMEGGIANI ANDREA ET AL: "Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics." THE JOURNAL OF BIOLOGICAL CHEMISTRY. 3 FEB 2006, vol. 281, no. 5, 3 February 2006 (2006-02-03), pages 2893-2900, XP002409415 ISSN: 0021-9258 *
SONG H ET AL: "Crystal Structure of Intact Elongation Factor EF-Tu from Escherichia coli in GDP Conformation at 2.05A Resolution" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 285, no. 3, 22 January 1999 (1999-01-22), pages 1245-1256, XP004449631 ISSN: 0022-2836 *
VOGELEY L ET AL: "Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu.GDP and aurodox." THE JOURNAL OF BIOLOGICAL CHEMISTRY. 18 MAY 2001, vol. 276, no. 20, 18 May 2001 (2001-05-18), pages 17149-17155, XP002409414 ISSN: 0021-9258 cited in the application *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021155459A1 (fr) * 2020-02-03 2021-08-12 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food Procédés améliorés de conception rationnelle et de production de biomolécules

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