Larry J. Ross

Slow Polymerization of Mycobacterium tuberculosis FtsZ

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

Thiazolobenzimidazole: Biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

A TIBO derivative, R82913, is a potent inhibitor of HIV-1 reverse transcriptase with heteropolymer templates.

R82913, (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (a TIBO derivative), inhibited the replication of thirteen different strains of HIV-1 in CEM cells with a median IC50 of 0.15 microM. The concentration of compound that killed 50% of the cells was much higher (46 microM), indicating that R82913 has a high selectivity index. R82913 was 20-fold more potent than AZT-TP in the inhibition of HIV-1 reverse transcriptase in an assay using a naturally occurring template (ribosomal RNA) that more accurately resembles native viral RNA than a synthetic homopolymer. With this template, R82913 inhibited HIV-1 reverse transcriptase with an ID50 (0.01 microM) that is equal to, or lower than, the IC50 for this compound in all of our cell culture assays (0.01-0.65 microM). R82913 has no effect on the replication of HIV-2 in CEM cells and does not inhibit the reverse transcriptase from this virus.

Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution.

The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.

A Novel Inhibitor of Mycobacterium tuberculosis Pantothenate Synthetase

Pantothenate synthetase (PS; EC 6.3.2.1), encoded by the panC gene, catalyzes the essential adenosine triphosphate (ATP)–dependent condensation of D-pantoate and β-alanine to form pantothenate in bacteria, yeast, and plants; pantothenate is a key precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). Because the enzyme is absent in mammals and both CoA and ACP are essential cofactors for bacterial growth, PS is an attractive chemotherapeutic target. An automated high-throughput screen was developed to identify drugs that inhibit Mycobacterium tuberculosis PS. The activity of PS was measured spectrophotometrically through an enzymatic cascade involving myokinase, pyruvate kinase, and lactate dehydrogenase. The rate of PS ATP utilization was quantitated by the reduction of absorbance due to the oxidation of NADH to NAD+ by lactate dehydrogenase, which allowed for an internal control to detect interference from compounds that absorb at 340 nm. This coupled enzymatic reaction was used to screen 4080 compounds in a 96-well format. This discussion describes a novel inhibitor of PS that exhibits potential as an antimicrobial agent.

Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase

The gene for dihydrofolate reductase of Mycobacterium tuberculosis was amplified by polymerase chain reaction (PCR) from M. tuberculosis H37Rv strain genomic DNA. The protein was expressed in inclusion bodies in high yield in Escherichia coli under the control of the T7 promoter. Active enzyme was obtained by refolding from guanidine HCl and after a single chromatography step the sample was > 99% homogeneous with a specific activity of approximately 15.5 micromol min(-1) mg(-1). Mass spectrometry analysis confirmed the expected mass of 17.6 kDa. Gel filtration of the enzyme indicated that it was a monomer. Steady-state kinetic parameters were determined and the effect of pH and KCl on the enzyme examined. Methotrexate and trimethoprim inhibited the enzyme.

Novel pyridopyrazine and pyrimidothiazine derivatives as FtsZ inhibitors.

A series of pyridopyrazine and pyrimidothiazine derivatives have been synthesized and their activity against FtsZ from Mycobacterium tuberculosis (Mtb) and in vitro antibacterial activity against Mtb H(37)Ra and Mtb H(37)Rv are reported. Certain analogs described herein showed moderate to good inhibitory activity.

Cell-Based and Biochemical Analysis of the anti-HIV Activity of Combinations of 3′-azido-3′-deoxythymidine and Analogues of TIBO

The toxicity of 3′-azido-3′-deoxythymidine (AZT) and the appearance of drug-resistant mutants in patients treated with AZT emphasizes the critical importance of the development of alternative strategies for the therapy of AIDS patients. Combination antiviral chemotherapy provides an attractive therapeutic strategy since the dose of the individual agents may be lowered to reduce toxicity and the use of two potent antiviral agents may limit the development of drug resistance. Two analogues of tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO) potently and selectively inhibit the replication of HIV-1 in cell culture. In combination with AZT, either of the two TIBO compounds, R82913 and R86183, was highly synergistic in cell culture against HIV-1. However, in biochemical enzyme inhibition assays, utilizing recombinant HIV-1 reverse transcriptase, synergy was not detected at the enzymatic level. These results suggest that one of these two known inhibitors of HIV-1 reverse transcriptase may have a secondary mechanism of action distinct from inhibition of the reverse transcriptase.

Structure-based virtual screening against SARS-3CLpro to identify novel non-peptidic hits

Abstract Severe acute respiratory syndrome is a highly infectious upper respiratory tract disease caused by SARS-CoV, a previously unidentified human coronavirus. SARS-3CLpro is a viral cysteine protease critical to the pathogen’s life cycle and hence a therapeutic target of importance. The recently elucidated crystal structures of this enzyme provide an opportunity for the discovery of inhibitors through rational drug design. In the current study, Gold docking program was utilized to conduct extensive docking studies against the target crystal structure to develop a robust and predictive docking protocol. The validated docking protocol was used to conduct a structure-based virtual screening of the Asinex Platinum collection. Biological evaluation of a screened selection of compounds was carried out to identify novel inhibitors of the viral protease.

A novel quinoline derivative that inhibits mycobacterial FtsZ.

High throughput phenotypic screening of large commercially available libraries through two NIH programs has produced thousands of potentially interesting hits for further development as antitubercular agents. Unfortunately, these screens do not supply target information, and further follow up target identification is required to allow optimal rational design and development of highly active and selective clinical candidates. Cheminformatic analysis of the quinoline and quinazoline hits from these HTS screens suggested a hypothesis that certain compounds in these two classes may target the mycobacterial tubulin homolog, FtsZ. In this brief communication, activity of a lead quinoline against the target FtsZ from Mycobacterium tuberculosis (Mtb) is confirmed as well as good in vitro whole cell antibacterial activity against Mtb H37Rv. The identification of a putative target of this highly tractable pharmacophore should help medicinal chemists interested in targeting FtsZ and cell division develop a rational design program to optimize this activity toward a novel drug candidate.