Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti-M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti-M. tuberculosis activity.

Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects such as microcephaly in newborns and neurological disease like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here we report the development of small non-nucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (“N-pocket”) located next to a putative hinge region between the thumb and the palm subdomains, that was originally described for dengue virus (DENV) RdRp. We first tested DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules and assessed their potency using both enzymatic and cell-based assays. The most potent compound has an IC50 value of 7.3 μM and inhibits ZIKV replication in a cell-based assay with an EC50 value of 24.3 μM. Importantly we report four high-resolution crystal structures detailing how these NNIs insert into the N-pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment and hydration of the N-pocket from ZIKV RdRp compared to DENV RdRp, that are crucial for the design of improved antiviral inhibitors against ZIKV. IMPORTANCE Zika virus belongs to the flavivirus family that comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The NS5 polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthetized inhibitors targeting the Zika virus NS5. We showed that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrate specific binding to an allosteric site within the polymerase called the N-pocket. This work paves the way for future structure-based design of potent compounds specifically targeting the ZIKV RNA polymerase activity.

Mycobacteria harbor two main degradative proteolytic machineries, the caseinolytic protease ClpP1P2 and a proteasome. We recently showed that Bortezomib inhibits ClpP1P2 and exhibits whole cell activity against Mycobacterium tuberculosis. Bortezomib, a dipeptide with a boronic acid warhead, is a human proteasome inhibitor approved for cancer therapy. The boronic acid warhead of the compound has been shown to drive potency against both the human proteasome and ClpP1P2 protease. Selectivity for the bacterial ClpP1P2 protease over the human proteasome is lacking but needs to be achieved to move this new anti-tuberculosis lead forward. In this study we explored whether an alternative warhead could influence Bortezomib's selectivity. We synthesized an analog containing a chloromethyl ketone instead of the boronic acid warhead and determined potencies against the bacterial and human enzymes. Surprisingly, the analog retained activity against mycobacterial ClpP1P2 and was active against the mycobacterial proteasome, but was devoid of activity against the human proteasome. Interrogation of a set of chloromethyl ketone peptides identified three additional compounds similarly inhibiting both ClpP1P2 and the proteasome in the bacteria while leaving the human proteasome untouched. Finally, we showed that these compounds display bactericidal activity against M. tuberculosis with cytotoxicity ranging from acceptable to undetectable. These results suggest that selectivity over the human proteasome is achievable. Selectivity, together with dual-targeting of mycobacterial ClpP1P2 and proteasome makes this new scaffold an attractive starting point for optimization.

3-Deazaneplanocin A (DzNep) is a potential epigenetic drug for the treatment of various cancers. DzNep has been reported to deplete histone methylations, including oncogenic EZH2 complex, giving rise to epigenetic modifications that reactivate many silenced tumor suppressors in cancer cells. Despite its promise as an anticancer drug, little is known about the structure-activity relationships of DzNep in the context of epigenetic modifications and apoptosis induction. In this study, a number of analogues of DzNep were examined for DzNep-like ability to induce synergistic apoptosis in cancer cells in combination with trichostatin A, a known histone deacetylase (HDAC) inhibitor. The structure-activity relationship data thus obtained provide valuable information on the structural requirements for biological activity. The studies identified three compounds that show similar activities to DzNep. Two of these compounds show good pharmacokinetics and safety profiles. Attempts to correlate the observed synergistic apoptotic activities with measured S-adenosylhomocysteine hydrolase (SAHH) inhibitory activities suggest that the apoptotic activity of DzNep might not be directly due to its inhibition of SAHH.