The Natural Products Group of LBC, NIDDK, is active in two areas of research, both of which seek to identify and understand the structural basis and mechanisms of action of inhibitors to (1.) mycothiol-dependent biosynthesis and detoxification, and (2.) HIV fusion. 1. Eukaryotes employ glutathione as the major small molecular weight thiol for maintaining a reducing intracellular environment and in protecting cells from oxidative stress and alkylating agents. In contrast, Gram-positive bacteria employ alternative small molecular weight thiols that are unique to particular bacterial groups. In the case of actinomycetes, which include the mycobacteria, mycothiol replaces glutathione and is thought to function similarly to glutathione in eukaryotes. Two key enzymes from Mycobacterium tubercuolsis (strain Rv37) that are involved in biosynthesis of mycothiol (rv1170 coding for AcCys-Ins deacetylase), and mycothiol-dependent detoxification (rv1082 coding for mycothiol-S-conjugate amidase, MCA), have been identified. Chemical and transposon mutants of these genes in both M. smegmatis and M. tuberculsosis have been shown to exhibit increased sensitivity to all first-line antituberculars with the exception of isoniazid, for which resistance is observed. During the past year we have continued studies on synthesis and mechanism of action of inhibitors of MCA, and are just starting work with AcGI deacetylase. Before embarking on these synthetic efforts, we had earlier completed a total synthesis of mycothiol bimane and both D- and L-inositol isomers of AcGI to establish absolute stereochemistry of substrates, and to characterize Mtb enzymes. Since those initial studies we are taking two approaches toward synthesis of inhibitors. The first follows on the work of our collaborators Knapp and co-workers, and makes use of cyclohexyl thioglycoside analogs which offer a tremendous advantage in that they do not require the laborious resolution steps for D/L-inositol mixtures through. Thus, we are currently synthesizing a small library of compounds that incorporate features from our natural product inhibitors described in earlier reports and those of the substrates. Once synthesized, the compounds will be tested for their inhibitory activity on MCA and AcGI deacetylase, as well as their effects on M. smegmatis and M. tuberculosis growth. The second approach to synthesizing inhibitors retains the GlcN-Ins pseudodisaccharide. This chemistry is close to being optimized. At the same time, this chemistry is being worked out with the future intent of making libraries of analogs via solid phase parallel synthesis. 2. In order for enveloped viruses to infect cells, membrane fusion must occur. In the case of HIV, the events leading to fusion include binding of the HIV envelope glycoprotein gp120 to the primary receptor CD4, and subsequent binding to the chemokine receptors CXCR4 or CCR5, which together facilitate insertion of the fusion peptide of gp41 (i.e. the transmembrane subunit of the HIV envelope protein) into the host cell membrane, ultimately leading to fusion. Current anti-retrovirals target HIV protease and reverse transcriptase (RT), and one fusion inhibitors has just received FDA approval this year (the C-peptide of gp41 known as T-20 or Fuzeon). While we have witnessed remarkable success in the design and development of protease and RT inhibitors, these drugs are often poorly tolerated and prohibitively expensive for much of the underdeveloped world. The second aspect of our research focuses on inhibitors of viral entry, and encompasses the following projects: (i.) identification of small-molecule inhibitors to HIV fusion, (ii.) determination of the structural basis of the potent fusion-blocking activity of cyanobacterial carbohydrate-binding proteins, and (iii.) the design of proteinaceous inhibitors and/or antigens of HIV fusion. Thus, during the past two years, we have isolated and completed total structure elucidations for nine different small molecules, all of which derived from marine sponges. These include the bishomoscalarane-type terpenes phyllolactones A-E, the pyridine alkaloids batzellins and makaluvamines, and the guanidine alkaloids crambescidin 826 and dehydrocrambine A. All of these inhibit HIV-1 Env-mediated fusion with IC50 values ranging from 0.5-30 micromolar in a cell-cell fusion assay. We have taken special interest in the tricyclic and pentacyclic guanidine alkaloids based on earlier reports by the SmithKline group that demonstrated that a related group of marine natural products known as batzelladines can inhibit gp120-CD4 binding. Experiments with crambescidins 800 and 826 in our laboratory indicate that these novel pentacyclic alkaloids can also block HIV fusion by binding to gp120 because delayed addition of crambescidins results in decreasing inhibition, consistent with a fusion inhibitor acting at early stages of the fusion process. In addition, when Env-bearing effector cells were pretreated with these alkaloids, followed by washing with media, more than 50% inhibition of fusion was still observed. On the other hand, pretreatment of target cells with crambescidins, followed by thorough washing, had no effect on fusion, demonstrating that the crambescidins bind to gp120. Studies are currently underway to map the binding site of crambescidins on gp120 using well-characterized anti-gp120 antibodies. A second ongoing project to develop fusion inhibitors that has the potential to shed new light on vaccine design, is that of engineered, gp41-derived trimeric fusion inhibitors. During the past year we have engineered two soluble, covalently-linked, trimeric polypeptides, N35CCG-N13 and N34CCG comprising only the internal trimeric coiled-coil of the ectodomain of HIV-1 gp41. Both trimers inhibit HIV-1 envelope (Env) mediated cell fusion at nanomolar concentrations by targeting the exposed C-terminal region of the gp41 ectodomain in the pre-hairpin intermediate state. The IC50s for N35CCG-N13 and N34CCG are ~15 and ~95 nM, respectively, in a quantitative vaccinia virus-based reporter gene assay for HIV-1 Env mediated cell fusion using Env from the T tropic strain LAV. Polyclonal antibodies were raised against N35CCG-N13 and a tightly binding fraction of anti-N35CCG-N13 inhibits T and M tropic HIV-1 Env-mediated cell fusion with respective IC50's of ~0.5 and ~1.5 microg/ml at 37 deg C. The tightly binding anti-N35CCG-N13 antibody fraction targets the exposed internal trimeric coiled-coil in the pre-hairpin intermediate state of gp41 in a manner analogous to peptides derived from the C-region of the gp41 ectodomain. The potency of the tightly binding anti-N35CCG-N13 antibody fraction in the fusion assay is comparable to that of the broadly neutralizing monoclonal antibody 2G12. These results indicate that N35CCG-N13 is a potential anti-HIV therapeutic agent and represents a suitable immunogen for the generation of neutralizing monoclonal antibodies targeted to the internal trimeric coiled-coil of gp41. The data on the tightly binding anti-N35CCG-N13 antibody fraction demonstrate that the internal trimeric coiled-coil of gp41 in the pre-hairpin intermediate state is accessible to antibodies and that access is not restricted by either antibody size or the presence of a kinetic barrier. Finally, we are continuing with our work on cyanobacterial proteins that potently inhibit diverse strains of HIV and are currently working on another novel cyanobacterial protein. Ongoing studies include establishing the precise epitope involved in carbohydrate recognition, determining binding affinities and stoichiometries of binding, mapping the binding site/s on gp120, and determining a high resolution 3-dimensional structure for this
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