Zika virus (ZIKV) is a flavivirus spread by daytime-active Aedes spp. mosquitoes such as A. aegypti and A. albopictus. Previously thought to be a mild infection, the latest ZIKV outbreak in the Americas is causally associated with more severe symptoms as well as severe birth defects, such as microcephaly. Currently no vaccine or antiviral exists. However, recent progress has demonstrated the viral NS2B/NS3 protease may be a suitable target for the development of small-molecule antiviral agents. To better understand the ZIKV protease, we expressed, purified, and characterized unlinked and linked NS2B/NS3 protease corresponding to an isolate from the recent outbreak in Puerto Rico. Unlinked ZIKV protease is more active and binds substrate with greater affinity than linked ZIKV protease. Therefore, we propose that unlinked ZIKV protease be used when evaluating or designing ZIKV protease inhibitors. Additionally, potent inhibitors of related viral proteases, like West Nile Virus and Dengue virus, may serve as advanced starting points to identify and develop ZIKV protease inhibitors.

A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4 and GI-1 norovirus proteases. Compound 19, containing a 6-membered ring at the P1 position and a reactive aldehyde warhead exhibited sub-micromolar replicon inhibition. Retaining the same peptidyl scaffold, several reactive warheads were tested for protease inhibition and norovirus replicon inhibition. Of the six that were synthesized and tested, compounds 42, 43, and 45 potently inhibited the protease in biochemical assay and GI-1 norovirus replicon in the nanomolar range.

Norovirus is the leading cause of acute gastroenteritis worldwide with a yearly reported 700 million cases driving a $60 billion global socioeconomic burden. With no United States Food and Drug Administration approved therapeutics and the chance for severe chronic infection and life-threatening complications, researchers have identified the protease as a potential target. However, drug development has focused on the norovirus GI.1 strain despite its accounting for less than 5% of all outbreaks. Our lab aims to change focus for norovirus drug design from GI.1 to the highly infective GII.4, responsible for more than 50% of all outbreaks worldwide. With the first published crystal structure of the norovirus GII.4 protease, we have identified several significant differences in the structure and active site that have hindered development of a potent inhibitor targeting the norovirus GII.4 protease. With these new insights, we have begun designing compounds that demonstrate increased inhibition of the clinically most relevant norovirus GII.4 strain.

Background The progression of castration‐resistant prostate cancer (CRPC) still relies on the function of androgen receptor (AR), achieved by evolving mechanisms to reactivate AR signaling under hormonal therapy. Histone deacetylase inhibitors (HDACis) disrupt cytoplasmic AR chaperone heat shock protein 90 (Hsp90) via HDAC6 inhibition, leading to AR degradation and growth suppression of prostate cancer (PCa) cells. However, current HDACis are not effective in clinical trials treating CRPC. Methods We designed hybrid molecules containing partial chemical scaffolds of AR antagonist enzalutamide (Enz) and HDACi suberoylanilide hydroxamic acid (SAHA) as new anti‐PCa agents. We previously demonstrated that Enz‐HDACi hybrid drug 2‐75 targets both AR and Hsp90, which inhibits the growth of Enz‐resistant C4‐2 cells. In the current study, we further investigate the molecular and cellular actions of 2‐75 and test its anti‐PCa effects in vivo. Results Compared with Enz, 2‐75 had greater AR antagonistic effects by decreasing the stability, transcriptional activity, and nuclear translocation of intracellular AR. In addition to inhibition of full‐length AR (FL AR), 2‐75 downregulated the AR‐V7 variant in multiple PCa cell lines. Mechanistic studies indicated that the AR affinity of 2‐75 retains the drug in the cytoplasm of AR + PCa cells and further directs 2‐75 to the AR‐associated protein complex, which permits localized effects on AR‐associated Hsp90. Further, unlike pan‐HDACi SAHA, the cytoplasm‐retaining property allows 2‐75 to significantly inhibit cytoplasmic HDAC6 with limited impact on nuclear HDACs. These selective cytoplasmic actions of 2‐75 overcome the unfavorable resistance and toxicity properties associated with classical AR antagonists, HDACis, and Hsp90 inhibitors. Finally, 2‐75 showed greater antitumor activities than Enz in vivo on SQ xenografts derived from LNCaP cells. Conclusions Novel therapeutic strategy using newly designed 2‐75 and related AR antagonist‐HDACi hybrid drugs has great potential for effective treatment of CRPC.

In order to understand the specificity of interactions between the components of multidrug-resistant (MDR) efflux pumps and how they are recruited/assembled, we analyzed the effect of C-terminal truncation, deletion, and peptide swapping on the stability and functionality of OprM in Escherichia coli. The efflux activity of OprM was not affected by removing up to 19 amino acid residues from the C-terminus, while depletion of more than 20 residues or disruption the ₄₆₃LGGG₄₆₆ motif diminished both the stability and activity of OprM. The replacement of the OprM C-terminus 23 residues with the corresponding part of TolC or VceC did not affect the stability and the functionality of OprM. Therefore, it is confirmed that the C-terminal ₄₆₃LGGG₄₆₆ motif is one of the crucial components for the stability of OprM and for the functionality of the OprM-VceAB chimeric pump in E.coli. The results also indicate that one residue substitution on the hairpin domain of the membrane fusion protein (MFP) VceA could suppress the null like mutations on the C-terminal modified OprM. This finding will be the direct genetic evidence that the C-terminal domain of outer efflux protein (OEP) is involved in the functional assembly of OEP-MFP.