Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: design, synthesis, biological evaluation, and cocrystal structure solution of P1 glutamine isosteric replacements.

The investigation of tripeptide aldehydes as reversible covalent inhibitors of human rhinovirus (HRV) 3C protease (3CP) is reported. Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrates, generate transition state models of P1-P1' amide cleavage, and propose novel tripeptide aldehydes. Glutaminal derivatives have limitations since they exist predominantly in the cyclic hemiaminal form. Therefore, several isosteric replacements for the P1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. These compounds were found to be potent inhibitors of purified HRV-14 3CP with Kis ranging from 0.005 to 0.64 microM. Several have low micromolar antiviral activity when tested against HRV-14-infected H1-HeLa cells. The N-acetyl derivative 3 was also shown to be active against HRV serotypes 2, 16, and 89. High-resolution cocrystal structures of HRV-2 3CP, covalently bound to compounds 3, 15, and 16, were solved. These cocrystal structures were analyzed and compared with our original HRV-14 3CP-substrate and inhibitor models.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 1. Michael acceptor structure-activity studies.

The structure-based design, chemical synthesis, and biological evaluation of peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds incorporate various Michael acceptor moieties and are shown to irreversibly bind to HRV serotype 14 3CP with inhibition activities (kobs/[I]) ranging from 100 to 600 000 M-1 s-1. These inhibitors are also shown to exhibit antiviral activity when tested against HRV-14-infected H1-HeLa cells with EC50's approaching 0.50 microM. Extensive structure-activity relationships developed by Michael acceptor alteration are reported along with the evaluation of several compounds against HRV serotypes other than 14. A 2.0 A crystal structure of a peptide-derived inhibitor complexed with HRV-2 3CP is also detailed.

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 2. Peptide structure-activity studies.

The structure-based design, chemical synthesis, and biological evaluation of various peptide-derived human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of an ethyl propenoate Michael acceptor moiety and a tripeptidyl binding determinant. The systematic modification of each amino acid residue present in the binding determinant as well as the N-terminal functionality is described. Such modifications are shown to provide irreversible HRV-14 3CP inhibitors with anti-3CP activities (kobs/[I]) ranging from 60 to 280 000 M-1 s-1 and antiviral EC50's which approach 0.15 microM. An optimized inhibitor which incorporates several improvements identified by the structure-activity studies is also described. This molecule displays very rapid irreversible inhibition of HRV-14 3CP (kobs/[I] = 800 000 M-1 s-1) and potent antiviral activity against HRV-14 in cell culture (EC50 = 0.056 microM). A 1.9 A crystal structure of an S-alkylthiocarbamate-containing inhibitor complexed with HRV-2 3CP is also detailed.

Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.

Design and synthesis of novel 6,7-imidazotetrahydroquinoline inhibitors of thymidylate synthase using iterative protein crystal structure analysis.

Antifolate inhibitors of thymidylate synthase (TS) have primarily been based on the structure of folic acid. This paper describes the identification and development of novel 6,7-imidazotetrahydroquinoline TS inhibitors by iterative ligand design, synthesis, and crystallographic analysis of protein-inhibitor complexes. Beginning with a high-resolution crystal structure of E. coli TS (TS, EC 2.1.1.45), an imidazotetrahydroquinoline inhibitor was designed de novo to occupy the folate binding pocket. Structural modifications of the initial compound 1h (Ki approximately 5 microM human/E. coli TS) were then made on the basis of feedback from additional cocrystal structures and activity data. An amino group in the 2-position of the imidazole was found to increase the potency of the series by 1-2 orders of magnitude. Other substitutions on the imidazole ring (1-CH3, 2-CH3, 2-NHCH3, 2-SCH3) generally led to weaker inhibition. Additional improvements in activity were obtained by modification of the substituents on the tetrahydroquinoline nitrogen, bringing the Ki of three of the compounds below 15 nM against the human TS enzyme. The compounds were tested for cytotoxicity and were shown to inhibit the growth of three tumor cell lines in vitro.