Modern computational chemistry and drug discovery: structure generating programs.

During 1996 and 1997, the first reports were disclosed of active enzyme inhibitors based entirely on novel structures created by de novo methods. De novo methods have also been used to modify and significantly improve the binding affinity of an HIV protease inhibitor. Work continues in the improvement of methods for the de novo design of compounds which fit and chemically complement a binding site. De novo algorithms that generate only synthetically feasible structures have also been reported. In addition, methods are being developed for the automatic computer generation of virtual molecular libraries which can be searched to identify molecules to match a pharmacophore or fit into a binding site.

A Src SH2 selective binding compound inhibits osteoclast-mediated resorption.

BACKGROUND: The observations that Src(-/-) mice develop osteopetrosis and Src family tyrosine kinase inhibitors decrease osteoclast-mediated resorption of bone have implicated Src in the regulation of osteoclast-resorptive activity. We have designed and synthesized a compound, AP22161, that binds selectively to the Src SH2 domain and demonstrated that it inhibits Src-dependent cellular activity and inhibits osteoclast-mediated resorption. RESULTS: AP22161 was designed to bind selectively to the Src SH2 domain by targeting a cysteine residue within the highly conserved phosphotyrosine-binding pocket. AP22161 was tested in vitro for binding to SH2 domains and was found to bind selectively and with high affinity to the Src SH2 domain. AP22161 was further tested in mechanism-based cellular assays and found to block Src SH2 binding to peptide ligands, inhibit Src-dependent cellular activity and diminish osteoclast resorptive activity. CONCLUSIONS: These results indicate that a compound that selectively inhibits Src SH2 binding can be used to inhibit osteoclast resorption. Furthermore, AP22161 has the potential to be further developed for treating osteoporosis.

Protein phosphorylation and signal transduction modulation: chemistry perspectives for small-molecule drug discovery.

Protein phosphorylation has been exploited by Nature in profound ways to control various aspects of cell proliferation, differentiation, metabolism, survival, motility and gene transcription. Cellular signal transduction pathways involve protein kinases, protein phosphatases, and phosphoprotein-interacting domain (e.g., SH2, PTB, WW, FHA, 14-3-3) containing cellular proteins to provide multidimensional, dynamic and reversible regulation of many biological activities. Knowledge of cellular signal transduction pathways has led to the identification of promising therapeutic targets amongst these superfamilies of enzymes and adapter proteins which have been linked to various cancers as well as inflammatory, immune, metabolic and bone diseases. This review focuses on protein kinase, protein phosphatase and phosphoprotein-interacting cellular protein therapeutic targets with an emphasis on small-molecule drug discovery from a chemistry perspective. Noteworthy studies related to molecular genetics, signal transduction pathways, structural biology, and drug design for several of these therapeutic targets are highlighted. Some exemplary proof-of-concept lead compounds, clinical candidates and/or breakthrough medicines are further detailed to illustrate achievements as well as challenges in the generation, optimization and development of small-molecule inhibitors of protein kinases, protein phosphatases or phosphoprotein-interacting domain containing cellular proteins.

Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption.

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.

Definition and display of steric, hydrophobic, and hydrogen-bonding properties of ligand binding sites in proteins using Lee and Richards accessible surface: validation of a high-resolution graphical tool for drug design.

The accessible surface, described by Lee and Richards (the L&R surface: J. Mol. Biol. 1971, 55, 379), has remarkably useful properties for displaying ligand-protein interactions. The surface is placed one van der Waals radius plus one probe radius away from the protein atoms. The ligands are displayed in skeletal form. With a suitable probe radius, those parts of the ligand in good van der Waals contact with the protein binding site are found superimposed on the L&R surface. Display of the surface using parallel contours therefore provides a very powerful guide for interactive drug design because only ligand atoms lying on or close to the surface are in low-energy contact. The ability of the surface to accurately display steric complementarity between ligands and proteins was optimized using data from small molecule crystal structures. The possibility of displaying the chemical specificity of the binding site was also investigated. The surface can be colored to give precise information about chemical specificity. Electrostatic potential, electrostatic gradient, and distance to hydrogen-bonding groups were tested as methods of displaying chemical specificity. The ability of these methods to describe the complementarity actually observed in the interior of proteins was compared. High-resolution crystal data for ribonuclease and trypsin was used. The environment surrounding extended peptide chains in the protein was treated as a virtual binding site. The peptide chain served as a virtual ligand. This large sample of experimental data was used to measure the correlation between type of ligand atom and the calculated property of the nearest binding site surface. The best correlation was obtained using hydrogen-bonding properties of the binding site. Using this parameter the surface could be divided into three separate zones representing the hydrophobic, hydrogen-bond-acceptor, and hydrogen-bond-donor properties of the binding site. The percentage of hydrophobic ligand atoms found to lie closest to the hydrophobic protein surface was 91%. The equivalent scores for ligand hydrogen-acceptor atoms and hydrogen-donor atoms found at the corresponding complementarity zone were 94% and 91%. The surface zones can be readily displayed using three colors. To test the method on real ligand/binding site interactions, nine thermolysin-inhibitor complexes of known structure were evaluated using the parameters and criteria derived from the protein-packing study and a correlation between complementary contacts and logarithm of potency was obtained which had an r2 of 0.99.(ABSTRACT TRUNCATED AT 400 WORDS)

Meta-substituted benzofused macrocyclic lactams as zinc metalloprotease inhibitors.

The design, synthesis, and biochemical profile of meta-substituted benzofused macrocyclic lactams are described. The meta-substituted benzofused macrocyclic lactams were designed to have a degree of flexibility allowing the amide bond to occupy two completely different conformations while maintaining sufficient rigidity to allow for strong interaction between enzyme and inhibitor. Using TFIT, a novel molecular superimposition program, it was shown that the meta analogs could be readily superimposed onto our ACE inhibitor template whereas no low-energy superimpositions of the ortho-substituted macrocycles could be found. The macrocycles were prepared by tethering aldehyde 1 derived from S-glutamic acid or S-aspartic acid to a meta-substituted phosphonium bromide 2. Homologation to a monocarboxylic acid methyl ester malonate followed by deprotection and cyclization gave the macrocyclic frame. Further manipulation gave the desired compounds. Unlike the ortho-substituted benzofused macrocyclic lactams described in the previous paper which are selective NEP inhibitors, the meta-substituted compounds are dual inhibitors of both NEP and ACE. The most potent member of this new series, compound 16a, inhibited both enzymes with an IC50 = 8 nM in NEP and 4 nM in ACE.

Ortho-substituted benzofused macrocyclic lactams as zinc metalloprotease inhibitors.

The design and preparation of ortho-substituted benzofused macrocyclic lactams are described. The benzofused macrocyclic lactams were designed as neutral endopeptidase 24.11 (NEP) inhibitors. Docking studies were carried out in a model of thermolysin (TLN) using the MACROMODEL and QXP modeling programs to select suitable ring sizes. These studies predicted that the 11-, 12-, and 13-membered ring macrocyclic lactams would be active in both enzymes TLN and NEP. Good predictability of experimental results, within this series, of binding to thermolysin and to a lesser extent to NEP was observed. A visual comparison, docked at the active site of TLN, is presented for thiorphan, a 10-membered ring macrocycle and an 11-membered ring benzofused macrocyclic lactam. Potent inhibition of both NEP and thermolysin was obtained. The 11-membered ring macrocycle 25a is the most potent inhibitor from this series of compounds (TLN IC50 = 68 nM; NEP IC50 = 0.9 nM). The effects of prodrug 44b administered at 10 mg/kg po on plasma atrial natriuretic peptide (ANP) levels in conscious rats was greater than 200% over a 4 h period.

Design and synthesis of an orally active macrocyclic neutral endopeptidase 24.11 inhibitor.

A potent macrocyclic inhibitor of neutral endopeptidase (NEP) 24.11 was designed using a computer model of the active site of thermolysin. This 10-membered ring lactam represents a general mimic for any hydrophobic dipeptide in which the two amino acid side chains bind to an enzyme in a contiguous orientation. The parent 10-membered ring lactam was synthesized and exhibited excellent potency as an NEP 24.11 inhibitor (IC50 = 3 nM). In order to improve oral bioavailability, various functionality was attached to the macrocycle. These modifications lead to CGS 25155, an orally active NEP 24.11 inhibitor that slows down the degradation of the cardiac hormone atrial natriuretic factor, producing a lowering of blood pressure in the DOCA-salt rat model of hypertension.

Discovery of potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70.

A series of 1,2,4-oxadiazole analogues has been shown to be potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70, a potential therapeutic target for immune suppression. These compounds typically are 200-400-fold more potent than the native, monophosphorylated tetrapeptide sequences. When compared with the high-affinity zeta-1-ITAM peptide (Ac-NQL-pYNELNLGRREE-pYDVLD-NH(2), wherein pY refers to phosphotyrosine) some of the best 1,2, 4-oxadiazole analogues are approximately 1 order of magnitude less active. This series of compounds displays an unprecedented level of selectivity over the closely related tyrosine kinase Syk, as well as other SH2-containing proteins such as Src and Grb2. Gel shift studies using a protein construct consisting only of C-terminal ZAP-70 SH2 demonstrate that these compounds can effectively engage this particular SH2 domain.

Identification of dipeptidyl nitriles as potent and selective inhibitors of cathepsin B through structure-based drug design.

Cathepsin B is a member of the papain superfamily of cysteine proteases and has been implicated in the pathology of numerous diseases, including arthritis and cancer. As part of an effort to identify potent, reversible inhibitors of this protease, we examined a series of dipeptidyl nitriles, starting with the previously reported Cbz-Phe-NH-CH(2)CN (19, IC(50) = 62 microM). High-resolution X-ray crystallographic data and molecular modeling were used to optimize the P(1), P(2), and P(3) substituents of this template. Cathepsin B is unique in its class in that it contains a carboxylate recognition site in the S(2)' pocket of the active site. Inhibitor potency and selectivity were enhanced by tethering a carboxylate functionality from the carbon alpha to the nitrile to interact with this region of the enzyme. This resulted in the identification of compound 10, a 7 nM inhibitor of cathepsin B, with excellent selectivity over other cysteine cathepsins.

Aspartic protease inhibitors designed from computer-generated templates bind as predicted.

[reaction: see text] Novel tripeptide-derived peptidomimetics 1, 7ab, and 8ab, inspired by templates generated by the structure-generating program GrowMol, were synthesized, shown to inhibit Rhizopus chinensis pepsin, and found by X-ray crystallography to bind to the enzyme in the GrowMol-predicted mode. Repetitive evaluation of the computer-generated templates for synthetic feasibility and optimal enzyme interactions led to the designed compounds.

Design and synthesis of unsymmetrical peptidyl urea inhibitors of aspartic peptidases.

[structure: see text] The design, synthesis, and enzyme inhibition of a new class of aspartic peptidase inhibitors is described. Unsymmetrical ureas were designed from computer-generated structures. Using mechanism-based and substrate-based design techniques, potent pepsin inhibitors were developed and the binding mode was established. Two X-ray crystal structures of enzyme-bound inhibitors revealed a new binding mode that is closely related to the computer-generated binding mode.

A rapid method for the computation, comparison and display of molecular volumes.

This paper presents a method for the rapid computation of approximate molecular van der Waals volumes and their subsequent display. The procedure relies on bit representation of individual volume elements that are mapped into an array that stores the total molecular volume. Our method differs from previously described algorithms in its use of bit-encoded templates that define atomic van der Waals radii. For each atom in the molecule, for which the volume is to be computed, the relevant template is mapped into a bit array with an offset corresponding to the appropriate atomic position. Bit-wise Boolean operations can be used for volume comparisons (e.g., common volume and excluded volume). An algorithm for the graphical display of the molecular surface encompassing the computed volumes is also described. The speed of the method enables users to perform volume computations in a reasonable period of time with VAX-class computers on molecules containing as many as several hundred atoms.

Flexible matching of test ligands to a 3D pharmacophore using a molecular superposition force field: comparison of predicted and experimental conformations of inhibitors of three enzymes.

A computer procedure TFIT, which uses a molecular superposition force field to flexibly match test compounds to a 3D pharmacophore, was evaluated to find out whether it could reliably predict the bioactive conformations of flexible ligands. The program superposition force field optimizes the overlap of those atoms of the test ligand and template that are of similar chemical type, by applying an attractive force between atoms of the test ligand and template which are close together and of similar type (hydrogen bonding, charge, hydrophobicity). A procedure involving Monte Carlo torsion perturbations, followed by torsional energy minimization, is used to find conformations of the test ligand which cominimize the internal energy of the ligand and the superposition energy of ligand and template. The procedure was tested by applying it to a series of flexible ligands for which the bioactive conformation was known experimentally. The 15 molecules tested were inhibitors of thermolysin, HIV-1 protease or endothiapepsin for which X-ray structures of the bioactive conformation were available. For each enzyme, one of the molecules served as a template and the others, after being conformationally randomized, were fitted. The fitted conformation was then compared to the known binding geometry. The matching procedure was successful in predicting the bioactive conformations of many of the structures tested. Significant deviation from experimental results was found only for parts of molecules where it was readily apparent that the template did not contain sufficient information to accurately determine the bioactive conformation.

Estimation of peptide hormone noncovalent binding site functionality from frequencies of interaction of amino acid side chains in proteins.

A method is described for estimating the amino acid side chain functional groups most likely to occur at a receptor site (or other protein-type binding site) given a particular peptide hormone sequence. The predictions are based on mutual affinities of amino acid side chains, as manifested in the frequencies of side chain contacts occurring in proteins. The relative merits of various measures of these affinities are discussed, and a new set of contact frequency data based on 36 proteins (coordinate data taken from the Brookhaven Protein Data Bank) is presented. Evaluation of predictions was carried out using subunit-subunit interfaces as models for peptide-receptor association; accuracy of guesses was found to be 3-4 times better than random, according to this measure. Computer programs for carrying out all requisite data manipulations are described.

QXP: powerful, rapid computer algorithms for structure-based drug design.

New methods for docking, template fitting and building pseudo-receptors are described. Full conformational searches are carried out for flexible cyclic and acyclic molecules. QXP (quick explore) search algorithms are derived from the method of Monte Carlo perturbation with energy minimization in Cartesian space. An additional fast search step is introduced between the initial perturbation and energy minimization. The fast search produces approximate low-energy structures, which are likely to minimize to a low energy. For template fitting, QXP uses a superposition force field which automatically assigns short-range attractive forces to similar atoms in different molecules. The docking algorithms were evaluated using X-ray data for 12 protein-ligand complexes. The ligands had up to 24 rotatable bonds and ranged from highly polar to mostly nonpolar. Docking searches of the randomly disordered ligands gave rms differences between the lowest energy docked structure and the energy-minimized X-ray structure, of less than 0.76 A for 10 of the ligands. For all the ligands, the rms difference between the energy-minimized X-ray structure and the closest docked structure was less than 0.4 A, when parts of one of the molecules which are in the solvent were excluded from the rms calculation. Template fitting was tested using four ACE inhibitors. Three ACE templates have been previously published. A single run using QXP generated a series of templates which contained examples of each of the three. A pseudo-receptor, complementary to an ACE template, was built out of small molecules, such as pyrrole, cyclopentanone and propane. When individually energy minimized in the pseudo-receptor, each of the four ACE inhibitors moved with an rms of less than 0.25 A. After random perturbation, the inhibitors were docked into the pseudo-receptor. Each lowest energy docked structure matched the energy-minimized geometry with an rms of less than 0.08 A. Thus, the pseudo-receptor shows steric and chemical complementarity to all four molecules. The QXP program is reliable, easy to use and sufficiently rapid for routine application in structure-based drug design.

Application of pattern recognition techniques to the analysis of protein crystal structure data. I. Characteristics of per-residue side chain contact frequency distributions.

A statistical study of amino acid side chain contact interactions was carried out using a data set based on 36 protein structures. For each type of amino acid, a distribution of per-residue inter-side-chain contacts was obtained, over the observed span of zero to 11 contacts per residue. Significant observations included the following: 1) The mean number of inter-side-chain contacts is proportional to side chain surface area with the exception of Lys and Arg. 2) The mean number of contacts was greater for amino acids in beta-sheet relative to alpha-helical regions. 3) The more polar or surface-loving amino acids exhibited non-normal distributions, whereas distributions for the non-polar or interior-loving amino acids fell within accepted limits of normality.