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Biomedical subjects

R S Bohacek

Publications and source records attributed to R S Bohacek.

At least 19 recordsLinked to original sources
PubMed2005

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.

Drug Design↗
PubMed2001

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.

Animals↗
PubMed2001

A novel phosphotyrosine mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp): exploitation in the design of nonpeptide inhibitors of pp60(Src) SH2 domain.

The novel phosphotyrosine (pTyr) mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60(c-Src) (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.

Animals↗
PubMed2001

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.

Aspartic Acid Endopeptidases↗
PubMed2001

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.

Animals↗
PubMed2001

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.

Actins↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

An efficient synthesis of a 4'-phosphonodifluoromethyl-3'-formyl-phenylalanine containing SRC SH2 ligand.

A CuBr-mediated, regioselective cross-coupling between methyl 2,5-diiodobenzoate (4) and [(diethoxyphosphinyl)difluoromethyl]zinc bromide is reported. Palladium-catalyzed incorporation of an amino acid side chain, followed by subsequent modifications resulted in the rapid construction of 2. Compound 2 was designed to engage Cys188 of the Src SH2 domain, however, this was not observed spectroscopically.

Binding Sites↗
PubMed1999

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.

Enzyme Inhibitors↗
PubMed1998

Synthesis of novel cyclic protease inhibitors using Grubbs olefin metathesis.

The unusual amino acid bishomoallylglycine was synthesized and used to form cyclic P3-P1 tripeptide inhibitors via a Grubbs olefin metathesis method. These compounds show micro- to nanomolar inhibition of Rhizopus chinensis pepsin and represent a new class of simplified aspartic protease inhibitors lacking P' residues.

Alkenes↗
PubMed1997

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.

Computer Simulation↗
PubMed1997

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.

Algorithms↗
PubMed1995

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.

Amino Acid Sequence↗