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Biomedical subjects

Lawrence C Kuo

Publications and source records attributed to Lawrence C Kuo.

26 records · Page 2Linked to original sources
PubMed2003

Design, synthesis, and biological evaluation of monopyrrolinone-based HIV-1 protease inhibitors.

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors [(-)-6, (-)-7, (-)-23, (+)-24] based upon the 3,5,5-trisubstituted pyrrolin-4-one scaffold is described. Use of a monopyrrolinone scaffold leads to inhibitors with improved cellular transport properties relative to the earlier inhibitors based on bispyrrolinones and their peptide counterparts. The most potent inhibitor (-)-7 displayed 13% oral bioavailability in dogs. X-ray structure analysis of the monopyrrolinone compounds cocrystallized with the wild-type HIV-1 protease provided valuable information on the interactions between the inhibitors and the HIV-1 enzyme. In each case, the inhibitors assumed similar orientations for the P2'-P1 substituents, along with an unexpected hydrogen bond of the pyrrolinone NH with Asp225. Interactions with the S2 pocket, however, were not optimal, as illustrated by the inclusion of a water molecule in two of the three inhibitor-enzyme complexes. Efforts to increase affinity by displacing the water molecule with second and third generation inhibitors did not prove successful. Lack of success with this venture is a testament to the difficulty of accurately predicting the many variables that influence and build binding affinity. Comparison of the inhibitor positions in three complexes with that of Indinavir revealed displacements of the protease backbones in the enzyme flap region, accompanied by variations in hydrogen bonding to accommodate the monopyrrolinone ring. The binding orientation of the pyrrolinone-based inhibitors may explain their sustained efficacy against mutant strains of the HIV-1 protease enzyme as compared to Indinavir.

Animals↗
PubMed2003

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

Adenosine↗
PubMed2003

Small, low nanomolar, noncovalent thrombin inhibitors lacking a group to fill the 'distal binding pocket'.

Use of a chlorophenoxyacetamide P1 group with a pyridinone acetamide P2/P3 gave an exceptionally potent thrombin inhibitor (K(i)=40 pM). Truncation of the molecule at the N-terminus gave unique, low nanomolar, non-covalent thrombin inhibitors which do not have a group to fill thrombin's 'distal binding pocket'. A co-crystal structure indicates the importance of an intramolecular hydrogen bond between the P1 side chain and P1/P2 amide link in this series.

Acetamides↗
PubMed2002

Synthesis and activity of novel HIV protease inhibitors with improved potency against multiple PI-resistant viral strains.

Substitution of the t-butylcarboxamide substituent in analogues of the HIV protease inhibitor (PI) Indinavir with a trifluoroethylamide moiety confers greater potency against both the wild-type (NL4-3) virus and PI-resistant HIV. The trifluoroethyl substituent also affords a slower clearance rate in vivo (dogs); however, this may be due to more potent inhibition of at least two P450 isoforms.

Animals↗
PubMed2002

Crystal structure of the Apo, unactivated insulin-like growth factor-1 receptor kinase. Implication for inhibitor specificity.

The x-ray structure of the unactivated kinase domain of insulin-like growth factor-1 receptor (IGFRK-0P) is reported here at 2.7 A resolution. IGFRK-0P is composed of two lobes connected by a hinge region. The N-terminal lobe of the kinase is a twisted beta-sheet flanked by a single helix, and the C-terminal lobe comprises eight alpha-helices and four short beta-strands. The ATP binding pocket and the catalytic center reside at the interface of the two lobes. Despite the overall similarity to other receptor tyrosine kinases, three notable conformational modifications are observed: 1) this kinase adopts a more closed structure, with its two lobes rotated further toward each other; 2) the conformation of the proximal end of the activation loop (residues 1121-1129) is different; 3) the orientation of the nucleotide-binding loop is altered. Collectively, these alterations lead to a different ATP-binding pocket that might impact on inhibitor designs for IGFRK-0P. Two molecules of IGFRK-0P are seen in the asymmetric unit; they are associated as a dimer with their ATP binding clefts facing each other. The ordered N terminus of one monomer approaches the active site of the other, suggesting that the juxtamembrane region of one molecule could come into close proximity to the active site of the other.

Adenosine Triphosphate↗
PubMed2002

A combinatorial library of indinavir analogues and its in vitro and in vivo studies.

A combinatorial library of 300HIV protease inhibitors has been synthesized. The library was screened against recombinant wild-type and mutant HIV-1 protease enzymes. The pharmacokinetics of the library was evaluated by dosing in dogs. Compounds that are notably more potent than indinavir and have favorable pharmacokinetic properties were identified.

Animals↗
PubMed2002

A collaborative screening program for the discovery of inhibitors of HCV NS2/3 cis-cleaving protease activity.

This report describes the development of a cell-based assay for high-throughput screening and detection of small-molecule inhibitors for hepatitis C virus (HCV) NS2/3 protease. The HCV NS2/3 protease is essential for the normal infectious cycle of HCV. Generation of a cell-based assay for this cis-acting viral protease involved reporter constructs in which the NS2/3 protease sequence was inserted between the ,B-lactamase (BLA) reporter and a ubiquitin-based destabilization domain. In stable cell lines, NS2/3 cis cleavage of the NS2/3-BLA fusion protein resulted in differential stability of the cleaved versus uncleaved BLA reporter, providing a robust readout for protease activity. BLA reporter activity was shown to be a function of NS2/3-specific protease activity, by using genetic mutants of the NS2/3 sequence. In addition, the cell-based assay was validated and screened in a 384-well format on a fully automated robotic platform where small-molecule inhibitors of NS2/3 protease activity were identified.

Automation↗