Conformationally constrained analogues of endogenous tripeptide inhibitors of zinc metalloproteinases.

Two diastereomeric furan-2-carbonylamino-3-oxohexahydroindolizino[8,7-b]indole carboxylates, highly constrained analogues of endogenous pyroglutamyl tripeptide inhibitors of snake venom endopeptidases, have been prepared as potential inhibitors of adamalysin II and matrix metalloproteinases. They proved to be inactive against adamalysin II and weak inhibitors of gelatinase A, gelatinase B, stromelysin 1 and human neutrophil collagenase. Evaluation of the mode of binding of the (2R,5S,11bR) isomer in the active site of adamalysin II suggests that the decrease of potency may be due to the reorientation of the acylamino chain in three of the heterocyclic nucleus, to a short contact at the entrance of the S'(1) hydrophobic cleft and to the loss of flexibility of the tetracyclic nucleus in the P'(1), P'(2) region of the inhibitor, which prevents optimal arrangement in the S'(1) specificity subsite.

Extended form of a retro-inverso peptide stabilized by beta-sheet unidirectional H-bonds: Crystallographic and NMR evidence.

The crystallographic investigation of the retro-inverso peptide Bz-S-gAla-R-mAla-NHPh reveals an extended backbone conformation where the NH groups of the gem-diamino alkyl moiety and the CO groups of the malonyl residue face side by side. This extended conformation, presenting all carbonyls on opposite sides of the NH groups, is stabilized by interstrand H-bonds running in a single direction of the parallel beta-sheets that characterize the crystal packing. These sheets differ from the beta-sheets formed by native amino acids only. (1)H-NMR nuclear Overhauser effect spectroscopy (NOESY) experiments suggest that a conformation similar to that found in the crystal also prevails in dimethylsulfoxide solution. Previous potential energy calculations of gem-diamino alkyl (g) and malonyl (m) Ala residues predicted that extended forms were less stable than the helical ones because of strong electrostatic repulsions between the parallel polar groups. Similar arguments were invoked to give more weight to helical forms of the retro-peptide units in the proposal of packing models of some nylons in their crystalline polar regions. The present findings show that both g and m Ala residues can experience the extended conformation in the beta-sheet aggregation. The energy increase occurring in one strand, due to the parallel orientation of consecutive peptide dipoles, is more than compensated by favorable cooperative interactions among head-to-tail aligned peptide dipoles of facing strands, resulting in the formation of two C==O...H==N H-bonds per residue.

Two crystal structures of human neutrophil collagenase, one complexed with a primed- and the other with an unprimed-side inhibitor: implications for drug design.

Two crystal structures of human neutrophil collagenase (HNC, MMP-8), one complexed with a primed- and the other with an unprimed-side inhibitor, were determined using synchrotron radiation at 100 K. Both inhibitors contain non-hydroxamate zinc-binding functions. The Pro-Leu-L-Trp(P)(OH)(2) occupies the unprimed region of the active site, furnishes new structural information regarding interaction between the catalytic zinc ion and the phosphonate group, and is the only example of occupation of the S(1) subsite of MMP-8 by the bulky tryptophan side chain. The (R)-2-(biphenyl-4-ylsulfonyl)-1,2,3, 4-tetrahydroisochinolin-3-carboxylic acid, a conformationally constrained D-Tic derivative, accommodates its biphenyl substituent into the deep primary specificity S(1)' subsite, inducing a widening of the entrance to this pocket; this modification of the protein, mainly consisting in a shift of the segment centered at Pro217, is observed for the first time in MMP-8 complexes. Cation-aromatic interactions can stabilize the formation of both complexes, and the beneficial effect of aromatic substituents in proximity of the catalytic zinc ion is discussed. The phosphonate group bound to either a primed- or unprimed-side inhibitor maintains the same relative position with respect to the catalytic zinc ion, suggesting that this binding function can be exploited for the design of combined inhibitors assembled to interact with both primed and unprimed regions of the active cleft.

Peptides containing the sulfonamide junction. 2. Structure and conformation of Z-Tau-Pro-D-Phe-NHiPr.

The taurine (Tau) containing N-protected pseudotripeptide isopropylamide Z-Tau-Pro-D-Phe-NHiPr (1) has been specifically designed and synthesized as suitable model to test the ability of the sulfonamido group to participate as H-bond acceptor to a type II beta-turn and to get information on the preferred rotameric conformation around the S-N bond and the hybridization state of the nitrogen atom. The present structural investigation reveals that, although the sulfonamide junction is invariably folded in a gauche mode, the beta-turn structure, stabilized by the 4 --> 1 hydrogen bond, is not found in the crystal and the sulfonamido oxygen atoms are not involved in any intra- or intermolecular hydrogen-bond interaction. More than one conformer populates the CDCl(3) solution with only a minor contribution by the expected beta-turn. The Pro nitrogen is significantly pyramidalized and the nitrogen lone pair points in opposite direction to that of the Pro C(alpha)H bond thus adopting R chirality, in an arrangement practically identical to that found in the previously studied homochiral analogue Z-Tau-Pro-Phe-NHiPr.

Inhibition of adamalysin II and MMPs by phosphonate analogues of snake venom peptides.

Phosphonate analogues of the peptidomimetic N-(Furan-2-yl)carbonyl-Leu-Trp-OH were prepared with the goal of evaluating the effect of phosphonate for carboxylate replacement on binding with snake venom metalloproteinases and MMPs. N-(Furan-2-yl)carbonyl-Leu-L-Trp(P)-(OH)2 showed a 75-fold increase of the inhibiting activity against adamalysin II, a snake venom metalloproteinase structurally related to MMPs and TACE. Both the phosphonate and carboxylate peptidomimetics fit into the active site adopting a retrobinding mode and provide the structural base for a new class of metalloproteinases inhibitors.

Synthesis, conformation, and biological activity of two fMLP-OMe analogues containing the new 2-[2'-(methylthio)ethyl]methionine residue.

The new C alpha-tetrasubstituted alpha-amino acid residue 2-[2'-(methylthio)ethyl]methionine (Dmt) has been introduced into the reference chemotactic tripeptide HCO-Met-Leu-Phe-OMe (fMLP-OMe) in place of the leucine or methionine, respectively. The biological activity of the new analogues [Dmt2]fMLP-OMe (2) and [Dmt1]fMLP-OMe (3) has been determined; whereas 2 is active toward human neutrophils, stimulating directed migration, superoxide anion generation, and lysozyme release, 3 results practically inactive in all tested assays. A conformational analysis on 2 and 3 has been performed in solution by using ir absorption and 1H-nmr. The conformation of 2 was also examined in the crystal by x-ray diffraction methods. Both 2 and 3 adopt fully extended conformation in correspondence with the Dmt residue. Biological and conformational results are discussed and compared with related previously studied models.

2 angstrom X-ray structure of adamalysin II complexed with a peptide phosphonate inhibitor adopting a retro-binding mode.

The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro-binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1'. An adamalysin-based model of tumor necrosis factor-alpha-converting enzyme (TACE) reveals a smaller S1' pocket for this enzyme.

Modified chemotactic peptides: synthesis, conformation, and activity of HCO-Thp-Ac6c-Phe-OMe.

HCO-Thp-Ac6c-Phe-OMe (3) has been synthesized as a new analogue of the prototypical chemotactic agent HCO-Met-Leu-Phe-OMe (fMLP-OMe). Compound 3 contains 4-aminotetra-hydrothiopyran-4-carboxylic acid (Thp) and 1-aminocyclohexane-1-carboxylic acid (Ac6c) as achiral, conformationally restricted mimics of Met and Leu, respectively. In the crystal, the formyltripeptide adopts an helical conformation at the Thp and Ac6c residues, of the type alpha R and alpha L, respectively, whereas the C-terminal phenylalanine is quasi-extended. A system of two consecutive gamma-turns, centered at the first two residues, better explains the nmr data as compared with an alternative beta-turn structure. The conformation of the new analogue 3 is compared with those of two related peptides containing Thp as N-terminal residue. The biological activity of 3 has been determined on human neutrophils and compared to that of the previously studied model [Ac6c2] fMLP-OMe. While the above analogue is highly active in the superoxide anion production, the new tripeptide 3 is practically unable to elicit any of the tested biological activities.

Sweet and bitter taste: structure and conformations of two aspartame dipeptide analogues.

The synthesis and X-ray diffraction analysis of two dipeptide taste ligands have been carried out as part of our study of the molecular basis of taste. The compounds L-aspartyl-D-alpha-methylphenylalanine methyl ester [L-Asp-D-(alpha Me)Phe-OMe] and L-aspartyl-D-alanyl-2,2,5, 5-tetramethylcyclopentanyl ester [L-Asp-D-Ala-OTMCP] elicit bitter and sweet taste, respectively. The C-terminal residues of the two analogues adopt distinctly different conformations in the solid state. The aspartyl moiety assumes the same conformation found in other dipeptide taste ligands with the side-chain carboxylate and the amino groups forming a zwitterionic ring with a conformation defined by psi, chi 1 = 157.7 degrees, -61.5 degrees for L-Asp-D-Ala-OTMCP and 151.0 degrees, -68.8 degrees for L-Asp-D-(alpha Me)Phe-OMe. In the second residue, a left-handed helical conformation is observed for the (alpha Me)Phe residue of L-Asp-D-(alpha Me)Phe-OMe with phi 2 = 49.0 degrees and psi 2 = 47.9 degrees, while the Ala residue of L-Asp-D-Ala-OTMCP adopts a semi-extended conformation characterized by dihedral angles phi 2 = 62.8 degrees and psi 2 = -139.9 degrees. The solid-state structure of the bitter L-Asp-D-(alpha Me)Phe-OMe is extended: while the crystal structure of the sweet L-Asp-D-OTMCP roughly adopts the typical L-shaped structure shown by other sweeteners. The data of L-Asp-D-(alpha Me)Phe-OMe are compared with those of its diastereoisomer L-Asp-L-(alpha Me)Phe-OMe. Conformational analysis of the two taste ligands in solution by NMR and computer simulations agrees well with our model for sweet and bitter tastes.

For-Met-Lys-Phe-For-Met-Lys-Phe-: a new cyclic analogue of the chemotactic formylpeptides.

As a continuation of the studies on chemotactic N-formylpeptides, we report here the synthesis and activity of a new cyclic analogue of the prototypical ligand For-Met-Leu-Phe-OMe. The new compound For-Met-Lys-Phe-For-Met-Lys-Phe- (4) contains a 20-membered cyclic moiety made up of a dimeric -Lys-Phe- sequence in which For-Met is attached to each Lys alpha-NH2 and hence remains outside the ring. The conformation in the crystal of the cyclic precursor of 4, namely Boc-Lys-Phe-Boc-Lys-Phe- (2) and the activity of the structurally related linear analogue For-Met-Lys(Z)-Phe-OBzl (6), have also been examined. The new analogues 4 and 6 are active as chemoattractants, secretagogues, and superoxide anion generating agents, when tested on human neutrophils. The structure-activity relationship is discussed and related to that of a previously studied cyclic model.

X-ray crystal structure, partitioning behavior, and molecular modeling study of piracetam-type nootropics: insights into the pharmacophore.

To detect possible molecular determinants of amnesia-reverting activity, the conformational properties of a number of rigid and flexible piracetam-type cognition enhancers have been assessed by X-ray diffraction, NMR spectroscopy, and ab initio and high-temperature-quenched molecular dynamics (QMD) calculations. The structures of the preferred conformers in solution derived from 1H-NMR spectral analysis were in good agreement with those found by QMD calculations. Interestingly, the calculation of the average molecular lipophilicity potential on the water-accessible surface of the selected conformers was helpful in interpreting the partitioning behavior observed by measuring octanol-water partition coefficients and capacity factors in reversed-phase high-performance liquid chromatography. While lipophilicity does not play a relevant role, the distance between polar groups, accounted for by the distance between carbonyl oxygens, emerges as a factor, among others, which should influence the amnesia-reversal activity of piracetam-type nootropics.

Modified chemotactic peptides: synthesis, conformation, and biological activity of For-Thp-Leu-delta ZPhe-OMe.

For-Thp-Leu-delta ZPhe-OMe (2), an analogue of the chemotactic tripeptide For-Met-Leu-Phe-OMe, containing 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and (Z)-2,3-didehydrophenylalanine (delta ZPhe) as achiral, conformationally restricted mimics of Met and Phe, respectively, has been synthesized. In the crystal the new formyltripeptide adopts a type I beta-turn conformation stabilized by a weak H bond between the formylic oxygen and the delta ZPhe NH. 1H-nmr analysis based on NH solvent accessibility and nuclear Overhauser effect experiments suggests that the beta-turn is not preferred in CDCl3 solution where a gamma-turn, centered at the Thp residue, prevails. The biological activity of 2 has been determined on human neutrophils and compared to that of previously studied analogues. The tripeptide 2 is practically unable to elicit superoxide anion production and lysozyme release, while slight, but not statistically significant activity was induced in chemotaxis. The role of the orientation of the aromatic ring with respect to the backbone adjacent atoms is discussed.

Modified chemotactic peptides: synthesis, crystal conformation, and activity of For-Hse(Me)-Leu-Phe-OMe.

The presence of the sulfur atom of the methionine side chain exerts significant effects at different levels on biochemical behavior of chemotactic N-formylpeptides. In order to acquire more information on this point, the synthesis, the conformation in the crystal, and the activity of For-Hse(Me)-Leu-Phe-OMe (2)--an oxygen analogue of For-Met-Leu-Phe-OMe (fMLP-OMe) containing the O-methyl-L-homoserine in place of the native methionine at position 1--is reported. The new analogue 2 adopts a conformation that is extended at the first two residues and folded at the C-terminal phenylalanine. This conformation is different from that of the parent fMLP-OMe and strikingly similar to that adopted by fMLP-OBu(t). The side-chain spatial orientation of 2 corresponds to that adopted by fMLP-OH when cocrystallized with an immunoglobulin possessing binding properties similar to those of neutrophil receptors. When tested on human neutrophils the formylpeptide 2 is more active than the parent in the stimulation of directed mobility and maintains both the granule enzyme release activity and the superoxide anion production.

Serine-containing 10-membered cyclodepsipeptides. Synthesis and molecular structure of PhCH2CO-DSer-Pro-Pro- and PhCH2CO-DSer-Pro-DPro-.

As a part of a research program aimed at studying synthesis and conformation of small ring peptides, the cyclization of diastereoisomeric N-phenylacetyl-seryl-propyl-proline tripeptides has been examined. Two 10-membered peptide lactones, PhCH2CO-DSer-Pro-Pro- 5a and PhCH2CO-DSer-Pro-DPro- 5b, have been isolated by treating the corresponding linear p-nitrophenyl esters with DBU in dry benzene. In these two compounds the serine lactone fragment (a common structural feature of several bioactive cyclodepsipeptides) is inserted into a highly strained small ring system. The conformation in the crystal of 5a and 5b has been studied by X-ray analysis. Both the 10-membered rings of 5a and 5b adopt an overall cis-cis-trans conformation in which the lactone junction is trans. The deviations from planarity of the peptide units vary from delta omega = 30 degrees for the DSer-Pro bond in 5b to delta omega = 5-6 degrees for the DSer-Pro bond in 5a and Pro-DPro bond in 5b. The skeletal atoms of 5b, containing the Pro-DPro sequence, are related by a pseudo-symmetry mirror plane passing through the Pro carbonyl and the opposite DSer C beta H2 group. In both the molecules the exocyclic amide bond adopts an extended conformation with respect to the DSer-Pro ring junction; this arrangement gives rise to a C5-type ring structure which is well evidenced in the case of 5a.

Synthesis, conformation, and activity of HCO-Met-delta Z Leu-Phe-OMe, an active analogue of chemotactic N-formyltripeptides.

In order to induce a beta-turn conformation into the chemotactic linear tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the new analogue N-formyl-L-methionyl-delta Z leucyl-L-phenylalanine methyl ester [delta Z Leu]2fMLP-OMe (1) has been synthesized. The conformational and biochemical consequences of this chemical modification have been determined. Analogue 1 has been synthesized by using N-carboxy-(Z)-alpha,beta-didehydroleucine anhydride as key compound to introduce the unsaturated residue at the central position of the tripeptide 1. The x-ray analysis shows that 1 adopts in the crystal a type II beta-turn conformation in which the new residue occupies the (i + 2) position, and an intramolecular H bond is formed between the formylic oxygen and the Phe NH. 1H-nmr analysis based on nuclear Overhauser effect measurements suggests that the same folded conformation is preferred in CDCl3 solution; this finding is also supported by molecular dynamics simulation. The biological activity of 1 has been determined on human neutrophils (polymorphonuclear leukocytes) and compared to that shown by fMLP-OMe. Chemotactic activity, granule enzyme release, and superoxide anion production have been determined. Analogue 1 is practically inactive as chemoattractant, highly active in the superoxide generation, and similar to the parent in the lysozyme release. The conformational restriction imposed on the backbone by the presence of the unsaturated residue is discussed in relation with the observed bioselectivity.

New chemotactic peptide analogs with high biological activity for human neutrophils.

As a part of a research programme aimed at studying structure activity relationships in the field of chemotactic peptides, modified analogs of the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) were examined for their capacity to activate several functions of human neutrophils. 4-Aminotetrahydrothiopyran-4carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) were chosen as achiral, conformationally restricted amino acids suitable for mimicking the external Met and Phe residues of FMLP-OMe. The replacement of both produces a high locomotion activity, greater than the parent peptide; in contrast, the two Thp-containing analogs induce neither superoxide production nor lysozyme release. From these results we can hypothesize two different signal transduction systems: one which provides for movement, the other for superoxide generation and granule enzyme release.

Synthesis and properties of chemotactic peptide analogs. II. HCO-Met-Leu-Phe-OMe analogs containing cyclic alpha,alpha-disubstituted amino acids as Met and Phe mimicking residues.

As a part of a research program aimed at studying structure activity relationship in the field of chemotactic peptides, modified analogs of the potent chemoattractant HCO-Met-Leu-Phe-OH (fMLP) of the general formula HCO-Xaa-Leu-Yaa-OMe are examined. 4-Aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) have been chosen as achiral, conformationally restricted amino acids suitable to mimick the external Met and Phe residues of fMLP-OMe. Studies on a first model, namely [Ain3]fMLP-OMe 1, have already been reported (12). Here the two remaining analogs [Thp1, Ain3] 2 and [Thp1] 3 have been synthesized. The conformation in the crystal of the disubstituted analog 2 has been determined and compared with those adopted by the parent fMLP-OMe and by previously studied models. The backbone conformation of 2 is characterized by helical folding centred at each of the three residues with the central Leu presenting helical handedness opposite to those of the two adjacent achiral residues. This conformation presents strong similarities with that adopted in the crystal by fMLP-OMe and resembles the conformation of fMLP bound to immunoglobulin (Bence-Jones dimer). The conformationally restricted analogs 2 and 3 are more active than the parent in the stimulation of directed mobility of human neutrophils but are practically inactive in the superoxide production. Crystals of 2 are orthorhombic, s.g. P2(1)2(1)2(1), with a = 21.934 (8), b = 10.856 (2), c = 10.380 (2) A. The structure has been refined to R = 0.071 for 2301 independent reflections with I greater than 1.5 sigma.

Ten-membered cyclotripeptides. III. Synthesis and conformation of cyclo(-Me beta Ala-Phe-Pro-) and cyclo(-Me beta Ala-Phe-DPro-).

The 10-membered cyclotripeptide cyclo(-Me beta Ala-Phe-Pro) 3 and its diastereoisomer cyclo(-Me beta Ala-Phe-DPro-) 4 have been synthesized under mild cyclization conditions starting from linear precursors containing C-terminal proline. The crystal and molecular structure of the two models has been determined by X-ray crystallography. Analysis of the NMR spectra supported by NOE data clearly indicates that the conformations found in the crystals are retained in solution. Both cyclotripeptides exhibit a cis-cis-trans backbone conformation. The two tertiary peptide bonds, at the proline and Me beta Ala nitrogen atoms, adopt a cis conformation whereas the CO-NH junctions are trans in both the models. The deviations from planarity of the peptide units vary from delta omega values of ca. 18 degrees for the Pro-Me beta Ala and DPro-Me beta Ala bonds to ca. 7 degrees for Phe-Pro and Phe-DPro bonds. Relevant conformational details of 3 and 4, as revealed by X-ray and NMR analysis, are reported. Crystals of 3 are monoclinic: P2(1), a = 5.317(2), b = 17.059(6), c = 9.514(3) A, beta = 99.18(3), Z = 2. The final R and Rw are 0.054 and 0.071 respectively. Crystals of 4 are orthorhombic: P2(1)2(1)2(1), a = 8.797(2), b = 19.440(9), c = 21.605(10) A, Z = 8. The final R and Rw are 0.069 and 0.104 respectively.

Synthesis and properties of chemotactic peptide analogs. I. Crystal structure and molecular conformation of HCO-Met-leu-Ain-OMe.

HCO-Met-Leu-Ain-OMe (2), an analog of the chemotactic peptide HCO-Met-Leu-Phe-OH, containing the conformationally blocked residue of the 2-aminoindane-2-carboxylic acid (Ain) has been synthesized and its crystal and molecular conformation has been determined. Crystals of 2 are monoclinic, space group P2(1), with a = 15.059(7), b = 18.548(7), c = 9.600(4) A; beta = 85.04(3) degrees. The structure has been solved by direct methods and refined to R = 0.069 for 2813 independent reflections with I greater than 2.5 sigma (I). Two independent molecules A and B have been found in the asymmetric unit of the crystal of 2. Their conformation can be described as extended at the Met and Leu residues, but folded at the C-terminal Ain residue. The helical folding is left- and right-handed in the A and B molecule, respectively. The crystal packing is characterized by ribbons of intermolecular hydrogen bonded molecules extended along the c direction. The constrained analog 2 is highly active in the superoxide production, thus indicating that a stabilization of a helical folding at the C-terminal region of chemotactic tripeptides maintains the activity. The orientation of the aromatic ring, with respect to its adjacent backbone atoms, does not seem critical for the activity.