Combining computational methods for hit to lead optimization in Mycobacterium tuberculosis drug discovery.

PURPOSE: Tuberculosis treatments need to be shorter and overcome drug resistance. Our previous large scale phenotypic high-throughput screening against Mycobacterium tuberculosis (Mtb) has identified 737 active compounds and thousands that are inactive. We have used this data for building computational models as an approach to minimize the number of compounds tested. METHODS: A cheminformatics clustering approach followed by Bayesian machine learning models (based on publicly available Mtb screening data) was used to illustrate that application of these models for screening set selections can enrich the hit rate. RESULTS: In order to explore chemical diversity around active cluster scaffolds of the dose-response hits obtained from our previous Mtb screens a set of 1924 commercially available molecules have been selected and evaluated for antitubercular activity and cytotoxicity using Vero, THP-1 and HepG2 cell lines with 4.3%, 4.2% and 2.7% hit rates, respectively. We demonstrate that models incorporating antitubercular and cytotoxicity data in Vero cells can significantly enrich the selection of non-toxic actives compared to random selection. Across all cell lines, the Molecular Libraries Small Molecule Repository (MLSMR) and cytotoxicity model identified ~10% of the hits in the top 1% screened (>10 fold enrichment). We also showed that seven out of nine Mtb active compounds from different academic published studies and eight out of eleven Mtb active compounds from a pharmaceutical screen (GSK) would have been identified by these Bayesian models. CONCLUSION: Combining clustering and Bayesian models represents a useful strategy for compound prioritization and hit-to lead optimization of antitubercular agents.

Structure of Mycobacterium tuberculosis FtsZ reveals unexpected, G protein-like conformational switches.

We report three crystal structures of the Mycobacterium tuberculosis cell division protein FtsZ, as the citrate, GDP, and GTPgammaS complexes, determined at 1.89, 2.60, and 2.08A resolution. MtbFtsZ crystallized as a tight, laterally oriented dimer distinct from the longitudinal polymer observed for alphabeta-tubulin. Mutational data on Escherichia coli FtsZ suggest that this dimer interface is important for proper protofilament and "Z-ring" assembly and function. An alpha-to-beta secondary structure conformational switch at the dimer interface is spatially analogous to, and has many of the hallmarks of, the Switch I conformational changes exhibited by G-proteins upon activation. The presence of a gamma-phosphate in the FtsZ active site modulates the conformation of the "tubulin" loop T3 (spatially analogous to the G-protein Switch II); T3 switching upon gamma-phosphate ligation is directly coupled to the alpha-to-beta switch by steric overlap. The dual conformational switches observed here for the first time in an FtsZ link GTP binding and hydrolysis to FtsZ (and tubulin) lateral assembly and Z-ring contraction, and they are suggestive of an underappreciated functional analogy between FtsZ, tubulin and G-proteins.

Redoxal, an inhibitor of de novo pyrimidine biosynthesis, augments APOBEC3G antiviral activity against human immunodeficiency virus type 1.

APOBEC3G (A3G) is a cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA; deamination-independent mechanisms are also implicated. HIV-1 Vif protein counteracts A3G by inducing its proteasomal degradation. Thus, the Vif-A3G axis is a potential therapeutic target. To identify compounds that inhibit Vif:A3G interaction, a 307,520 compound library was tested in a TR-FRET screen. Two identified compounds, redoxal and lomofungin, inhibited HIV-1 replication in peripheral blood mononuclear cells. Lomofungin activity was linked to A3G, but not pursued further due to cytotoxicity. Redoxal displayed A3G-dependent restriction, inhibiting viral replication by stabilizing A3G protein levels and increasing A3G in virions. A3G-independent activity was also detected. Treatment with uridine or orotate, intermediates of pyrimidine synthesis, diminished redoxal-induced stabilization of A3G and antiviral activity. These results identify redoxal as an inhibitor of HIV-1 replication and suggest its ability to inhibit pyrimidine biosynthesis suppresses viral replication by augmenting A3G antiviral activity.