Mark D. Shenderovich

Design of peptides, proteins, and peptidomimetics in chi space

Peptide and protein biological activities depend on their three dimensionals structures in the free state and when interacting with their receptors/acceptors. The backbone conformations such as alpha-helix, beta-sheet, beta-turn, and so forth provide critical templates for the three-dimensional structure, but the overall shape and intrinsic stereoelectronic properties of the peptide or protein important for molecular recognition, signal transduction, enzymatic specificity, immunomodulation, and other biological effects depend on arrangement of the side chain groups in three-dimensional chi space (their chi 1, chi 2, etc. torsional angles). In this paper we explore approaches to the de novo design of polypeptides and peptidomimetics with biased or specific conformational/topographical properties in chi space. We consider computational and experimental methods that can be used to examine the effects of specific structural modifications in constraining side chain groups of amino acid residues and their similarities in chi space to the natural amino acids to evaluate what sort of mimetics are likely to mimic normal amino acids. We then examine some of the asymmetric synthetic methods that are being developed to obtain the amino acid mimetics. Finally, we consider selected examples in the literature where these specialized amino acids have been incorporated in biologically active peptides and the specific insights they have provided regarding the topographical requirements for bioactive peptide potency, selectivity, and other biochemical and pharmacological properties. Constraints in chi space show great promise as useful tools in peptide, protein, and peptidomimetic de novo design of structures and pharmacophores with specific stereostructural, biochemical and biological properties.

Exploration of the conformational space of oxytocin and arginine-vasopressin using the electrostatically driven Monte Carlo and molecular dynamics methods

Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine-vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration-shell model, and by simulated-annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low-energy conformations obtained for both hormones were classified using the minimal-tree clustering algorithm and characterized according to the locations of beta-turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a beta-turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a beta-turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The latter difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x-ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr-derived distance constraints. Generally, the low-energy conformations obtained with the hydration-shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low-energy conformations do not satisfy all of the nmr-derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with beta-turns at residues 2,3, 3,4, and 4,5. The low-energy structures of OT with a beta-turn at residues 2,3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M. D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921-927], although the native hormone differs from the bicyclic analogue by the conformation of the C-terminal tripeptide. This finding confirms the hypothesis of different receptor-bound conformations of agonists and antagonists of OT.

Structural analysis of an HIV-1 protease I47A mutant resistant to the protease inhibitor lopinavir

We have identified a rare HIV‐1 protease (PR) mutation, I47A, associated with a high level of resistance to the protease inhibitor lopinavir (LPV) and with hypersusceptibility to the protease inhibitor saquinavir (SQV). The I47A mutation was found in 99 of 112,198 clinical specimens genotyped after LPV became available in late 2000, but in none of 24,426 clinical samples genotyped from 1998 to October 2000. Phenotypic data obtained for five I47A mutants showed unexpected resistance to LPV (86‐ to >110‐fold) and hypersusceptibility to SQV (0.1‐ to 0.7‐fold). Molecular modeling and energy calculations for these mutants using our structural phenotyping methodology showed an increase in the binding energy of LPV by 1.9–3.1 kcal/mol with respect to the wild type complex, corresponding to a 20‐ to >100‐fold decrease in binding affinity, consistent with the observed high levels of LPV resistance. In the WT PR–LPV complex, the Ile 47 side chain is positioned close to the phenoxyacetyl moiety of LPV and its van der Waals interactions contribute significantly to the ligand binding. These interactions are lost for the smaller Ala 47 residue. Calculated binding energy changes for SQV ranged from −0.4 to −1.2 kcal/mol. In the mutant I47A PR–SQV complexes, the PR flaps are packed more tightly around SQV than in the WT complex, resulting in the formation of additional hydrogen bonds that increase binding affinity of SQV consistent with phenotypic hypersusceptibility. The emergence of mutations at PR residue 47 strongly correlates with increasing prescriptions of LPV (Spearman correlation rs=0.96, P<.0001).

Structure‐based phenotyping predicts HIV‐1 protease inhibitor resistance

Mutations in HIV‐1 drug targets lead to resistance and consequent therapeutic failure of antiretroviral drugs. Phenotypic resistance assays are time‐consuming and costly, and genotypic rules‐based interpretations may fail to predict the effects of multiple mutations. We have developed a computational procedure that rapidly evaluates changes in the binding energy of inhibitors to mutant HIV‐1 PR variants. Models of WT complexes were produced from crystal structures. Mutant complexes were built by amino acid substitutions in the WT complexes with subsequent energy minimization of the ligand and PR binding site residues. Accuracy of the models was confirmed by comparison with available crystal structures and by prediction of known resistance‐related mutations. PR variants from clinical isolates were modeled in complex with six FDA‐approved PIs, and changes in the binding energy (ΔEbind) of mutant versus WT complexes were correlated with the ratios of phenotypic 50% inhibitory concentration (IC50) values. The calculated ΔEbind of five PIs showed significant correlations (R2 = 0.7–0.8) with IC50 ratios from the Virco Antivirogram assay, and the ΔEbind of six PIs showed good correlation (R2 = 0.76–0.85) with IC50 ratios from the Virologic PhenoSense assay. ΔEbind cutoffs corresponding to a four‐fold increase in IC50 were used to define the structure‐based phenotype as susceptible, resistant, or equivocal. Blind predictions for 78 PR variants gave overall agreement of 92% (kappa = 0.756) and 86% (kappa = 0.666) with PhenoSense and Antivirogram phenotypes, respectively. The structural phenotyping predicted drug resistance of clinical HIV‐1 PR variants with an accuracy approaching that of frequently used cell‐based phenotypic assays.

A three-dimensional model of the δ-opioid pharmacophore: Comparative molecular modeling of peptide and nonpeptide ligands

A comparative molecular modeling study of delta-opioid ligands was performed under the assumption that potent peptide and nonpeptide agonists may have common three-dimensional (3D) arrangement of pharmacophore groups upon binding to the delta-receptor. Low-energy conformations of the agonists 7-spiroindanyloxymorphone (SIOM) and 2-methyl-4a-alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12, 12a-alpha-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67), and a partial agonist oxomorphindole (OMI) were determined by high-temperature molecular dynamics (MD). A good spatial overlap was found for the pharmacophore groups of SIOM, TAN-67, and OMI, including the basic nitrogen, phenol hydroxyl, and two aromatic ring. Based on this overlap we proposed a 3D pharmacophore model for nonpeptide delta-opioid agonists with a distance of 7.0 +/- 1.3 A between the two aromatic rings and of 8.2 +/- 1.0 A between the nitrogen and phenyl ring. The potent and highly delta-opioid receptor selective agonist [(2S,3R)-TMT(1)]DPDPE, which shares global backbone constraints of the 14-membered disulfide cycle and a strong preference for the trans rotamer of the TMT(1) side chain, was chosen as a peptide template of the delta-opioid pharmacophore. Extensive MD simulations at 300 K with the AMBER force field were performed for [(2S,3R)-TMT(1)]DPDPE and the less potent [(2S, 3S)-TMT(1)]DPDPE analogue. Multiple MD trajectories were collected for each peptide starting from the x-ray structures of DPDPE and [L-Ala(3)]DPDPE and from models proposed in the literature. Low-energy MD conformations were filtered by the nonpeptide pharmacophore query and then directly superimposed with SIOM, OMI, and TAN-67. Two conformers of [(2S,3R)-TMT(1)]DPDPE that showed the best overlap with the nonpeptide pharmacophore (rms deviation </= 1. 0 A for N,O atoms and centroids of two aromatic rings) were selected as possible delta-receptor binding conformations. These conformations have similar backbone structures, and trans rotamers of the TMT(1) side-chain group. They are reasonably close to the crystal structure of [L-Ala(3)]DPDPE, and differ significantly from the crystal structure of DPDPE. The conformer with a gauche(-) rotamer of Phe(4) is most consistent with structure-activity relationships of delta-opioid peptides. The proposed 3D models were used for rational design of new nonpeptide delta-receptor ligands.

Discovery of (2S)-1-[4-(2-{6-amino-8-[(6-bromo-1,3-benzodioxol-5-yl)sulfanyl]-9H-purin-9-yl}ethyl)piperidin-1-yl]-2-hydroxypropan-1-one (MPC-3100), a purine-based Hsp90 inhibitor.

Modulation of Hsp90 (heat shock protein 90) function has been recognized as an attractive approach for cancer treatment, since many cancer cells depend on Hsp90 to maintain cellular homeostasis. This has spurred the search for small-molecule Hsp90 inhibitors. Here we describe our lead optimization studies centered on the purine-based Hsp90 inhibitor 28a containing a piperidine moiety at the purine N9 position. In this study, key SAR was established for the piperidine N-substituent and for the congeners of the 1,3-benzodioxole at C8. These efforts led to the identification of orally bioavailable 28g that exhibits good in vitro profiles and a characteristic molecular biomarker signature of Hsp90 inhibition both in vitro and in vivo. Favorable pharmacokinetic properties along with significant antitumor effects in multiple human cancer xenograft models led to the selection of 28g (MPC-3100) as a clinical candidate.

Analogues of 4-[(7-Bromo-2-methyl-4-oxo-3H-quinazolin-6-yl)methylprop-2-ynylamino]-N-(3-pyridylmethyl)benzamide (CB-30865) as Potent Inhibitors of Nicotinamide Phosphoribosyltransferase (Nampt)

We have shown previously that the target of the potent cytotoxic agent 4-[(7-bromo-2-methyl-4-oxo-3H-quinazolin-6-yl)methyl-prop-2-ynylamino]-N-(3-pyridylmethyl)benzamide (CB38065, 1) is nicotinamide phosphoribosyltransferase (Nampt). With its cellular target known we sought to optimize the biochemical and cellular Nampt activity of 1 as well as its cytotoxicity. It was found that a 3-pyridylmethylamide substituent in the A region was critical to cellular Nampt activity and cytotoxicity, although other aromatic substitution did yield compounds with submicromolar enzymatic inhibition. Small unsaturated groups worked best in the D-region of the molecule, with 3,3-dimethylallyl providing optimal potency. The E region required a quinazolin-4-one or 1,2,3-benzotriazin-4-one group for activity, and many substituents were tolerated at C² of the quinazolin-4-one. The best compounds showed subnanomolar inhibition of Nampt and low nanomolar cytotoxicity in cellular assays.

The stereochemical requirements of the novel δ-opioid selective dipeptide antagonist TMT-Tic

Abstract Five conformationally constrained dipeptide TMT- L -Tic analogues have been synthesized and evaluated for their bioactivity using in vitro bioassays. The most potent and selective analogue (2 S ,3 R )-TMT- L -Tic showed 9 nM binding affinity and 4000-fold selectivity for the δ vs μ opioid receptor. The lowest-energy conformation of (2 S ,3 R )-TMT- L -Tic is suggested to be bioactive one in which the χ 1 torsional angle is trans for TMT and gauche (+) for Tic.

Lead optimization of purine based orally bioavailable Mps1 (TTK) inhibitors.

Efforts to optimize biological activity, novelty, selectivity and oral bioavailability of Mps1 inhibitors, from a purine based lead MPI-0479605, are described in this Letter. Mps1 biochemical activity and cytotoxicity in HCT-116 cell line were improved. On-target activity confirmation via mechanism based G2/M escape assay was demonstrated. Physico-chemical and ADME properties were optimized to improve oral bioavailability in mouse.

Conformational analysis of β-methyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin by NMR spectroscopy and conformational energy calculations

Solution conformations of beta-methyl-para-nitrophenylalanine4 analogues of the potent delta-opioid peptide cyclo[D-Pen2, D-Pen5]enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and 3JHNC alpha H coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in DMSO were compared with low energy conformers obtained by energy minimization in the Empirical Conformational Energy Program for Peptides (ECEPP/2) force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homonuclear (3JH alpha H beta) and heteronuclear (3JH alpha C gamma) coupling constants and 13C chemical shifts, show that the beta-methyl substituent eliminates one of the three staggered rotamers of the torsion angle chi 1 for each stereoisomer of the beta-Me-p-NO2Phe4. Similar solution conformations were suggested for the L-Phe4-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe4-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main delta-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in the delta-receptor-bound state may assume a conformation different from their favorable conformations in DMSO.