Michael J. Otto

Synthesis and anti-HIV activity of pyrrolo-[1,2-d]-(1,4)-benzodiazepin-6-ones

Abstract The synthesis of novel pyrrolo annulated 1,4-benzodiazepines is described. These pyrrolo[1,2-d]-(1,4)-benzodiazepines have been found to have antiviral activity against HIV-1. Like other non nucleoside HIV-1 RT inhibitors, these compounds appear to be specific for HIV-1.

Multiple Mutations in the Human Immunodeficiency Virus Protease Gene Are Responsible for Decreased Susceptibility to Protease Inhibitors

The protease (PR) of the human immunodeficiency virus (HIV) is essential for replication of the virus, and accordingly has become an attractive target for the development of an antiretroviral drug. We have previously reported that passage of HIV-1 in the presence of increasing concentrations of the C-2 symmetrical, linear diol P9941 resulted in the isolation of virus with a valine-to-alanine change at position 82 (V82A) of the PR, and reduced sensitivity to certain PR inhibitors. In this study, we passaged four different variants of HIV-1 in increasing concentrations of XM323, and isolated variants with reduced sensitivity to inhibitors of PR. Twenty-three passages of HIV-1 (RF) in the presence of XM323 resulted in a variant that exhibited an approximately 100-fold reduction in susceptibility to XM323 and that contained V82F and I84V changes. When two other viruses, HIV-1 (RF41D2) and HIV-1(RF41E4), previously derived from HIV-1 (RF) by passage in the presence of P9941, were passaged in the presence of XM323, variants with V82A/L97V and M46L/V82A/L97V changes, respectively, were obtained. The M46L/V82A/L97V variant showed a 6-fold reduction in sensitivity to XM323, whereas the susceptibility of the V82A/L97V mutant remained unchanged. Seventeen passages of a clinical isolate of HIV-1, HIV-1 (Pat.E), in the presence of XM323 produced a V82F/L97V mutant with an approximately 9-fold reduction in sensitivity to XM323.

Identification of a clinical isolate of HIV-1 with an isoleucine at position 82 of the protease which retains susceptibility to protease inhibitors

The HIV-1 protease (PR) is essential for the production of mature virions. As such, it has become a target for the development of anti-HIV chemotherapeutics. Multiple passages of virus in cell culture in the presence of PR inhibitors have resulted in the selection of variants with decreased sensitivity to inhibitors of the PR. The most common alteration observed is a single amino acid change at position 82. This particular position has been well characterized by several laboratories as being important for the susceptibility of the virus to inhibitors of PR function. Mutations which result in the substitution of the wild-type valine with alanine, phenylalanine, threonine or isoleucine at position 82 of the PR have been associated with decreased sensitivity to several PR inhibitors. We describe here a clinical strain of HIV-1 that contains an isoleucine at position 82 of the PR instead of the usual valine. This strain is unique in that it was isolated from a patient that was anti-retroviral naive, and in the past, variants at position 82 of the PR have only been found after treatment of patients or cell culture with PR inhibitors. Moreover, this virus remains sensitive to PR inhibitors of the cyclic urea and C-2 symmetrical diol classes.

Inhibitors of picornavirus uncoating as antiviral agents

The discovery of the antiviral drugs known today can be attributed primarily to the tenacity of researchers and the serendipity of circumstances. Their development, however, has been carefully orchestrated by intense study of the mode of action of these compounds and increased understanding of molecular events involved in virus replication. We are presently entering a new era of development in antiviral chemotherapy characterized by the synthesis of chemicals designed to inhibit specific viral targets. One target of chemical interference is the process of viral uncoating, an essential step in viral replication which results in the release of the viral genome into either the cytoplasm (RNA viruses) or nucleus (DNA viruses) of the cell. Our goal has been to synthesize and develop compounds which inhibit the uncoating process of picornaviruses. If successful, this could result in the identification of compounds effective in the treatment of diseases caused by the enteroand rhinovirus members of the picornavirus family, including the causative agents of aseptic meningitis, poliomyelitis and the common cold.

Common cold viruses

Abstract The structure of a human common cold (rhinovirus) serotype, human rhinovirus 14 (HRV14) is providing extensive information on viral assembly, stability, neutralization by antibodies and antiviral agents as well as the site of receptor attachment.

In vitro susceptibility of clinical isolates of HIV-1 to XM323, a non-peptidyl HIV protease inhibitor.

ObjectiveTo determine the in vitro susceptibility of primary clinical isolates and laboratory strains of HIV-1 to XM323. MethodsThe AIDS Clinical Trials Group/US Department of Defense p24 antigen-based consensus assay was used to determine in vitro susceptibility of 57 primary clinical isolates and three laboratory strains of HIV-1 to XM323, zidovudine, zalcitabine (ddC), and didanosine (ddl). ResultsThe concentrations of compound required to inhibit viral p24 antigen production by 50% [median inhibitory concentration (IC50)] for nucleosides were as follows: zidovudine, 0.001->5μM; ddC, < 0.01–0.23μM; ddl, 0.2-25μM). Against both nucleoside susceptible and resistant isolates XM323 exhibited potent inhibition with IC50 values of < 0.02–0.27 μM and IC90 values of 0.03–1.17 μM. ConclusionsXM323 is a potent inhibitor of diverse clinical isolates of HIV-1 in vitro and represents a novel class of non-peptidyl inhibitors of HIV-1 protease.

Molecular Characterization of HIV-2 (ROD) Protease Following PCR Cloning from Virus Infected H9 Cells

A 450 nucleotide sequence corresponding to the nucleotides 1931-2380 of the viral genome (8) was amplified by polymerase chain reaction (PCR) using template DNA prepared from HIV-2 (ROD) infected H9 cells. The sequence codes for HIV-2 protease and its N-terminal flanking peptide. An identical DNA sequence was obtained from three independent PCR amplifications, which differs from the published sequence of HIV-2 (ROD) in 7 nucleotides scattered throughout the region of the cloned DNA. The cloned DNA was expressed in E. coli cells and resulted in the synthesis of a correctly processed HIV-2 protease, which is enzymatically active. Therefore, none of the seven nucleotide changes, which resulted in two amino acid substitutions, affect the autoproteolytic or trans-cleaving activities of the HIV-2 protease.