Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer.

Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4's hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.

Targeting Mycobacterium tuberculosis CoaBC through Chemical Inhibition of 4'-Phosphopantothenoyl-l-cysteine Synthetase (CoaB) Activity.

Coenzyme A (CoA) is a ubiquitous cofactor present in all living cells and estimated to be required for up to 9% of intracellular enzymatic reactions. Mycobacterium tuberculosis (Mtb) relies on its own ability to biosynthesize CoA to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the pathway to CoA biosynthesis is recognized as a potential source of novel tuberculosis drug targets. In prior work, we genetically validated CoaBC as a bactericidal drug target in Mtb in vitro and in vivo. Here, we describe the identification of compound 1f, a small molecule inhibitor of the 4'-phosphopantothenoyl-l-cysteine synthetase (PPCS; CoaB) domain of the bifunctional Mtb CoaBC, and show that this compound displays on-target activity in Mtb. Compound 1f was found to inhibit CoaBC uncompetitively with respect to 4'-phosphopantothenate, the substrate for the CoaB-catalyzed reaction. Furthermore, metabolomic profiling of wild-type Mtb H37Rv following exposure to compound 1f produced a signature consistent with perturbations in pantothenate and CoA biosynthesis. As the first report of a direct small molecule inhibitor of Mtb CoaBC displaying target-selective whole-cell activity, this study confirms the druggability of CoaBC and chemically validates this target.

Compartmentation of ß2 -adrenoceptor stimulated cAMP responses by phosphodiesterase types 2 and 3 in cardiac ventricular myocytes.

BACKGROUND AND PURPOSE: In cardiac myocytes, cyclic AMP (cAMP) produced by both ß1 - and ß2 -adrenoceptors increases L-type Ca2+ channel activity and myocyte contraction. However, only cAMP produced by ß1 -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of ß2 -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation. EXPERIMENTAL APPROACH: The cAMP responses produced by ß1 -and ß2 -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex. KEY RESULTS: Selective activation of ß1 - or ß2 -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of ß1 -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of ß2 -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4. CONCLUSION AND IMPLICATIONS: These results support the conclusion that cAMP produced by ß2 -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site.

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

Hepatic histopathology and clinical characteristics associated with antiretroviral therapy in HIV patients without viral hepatitis.

BACKGROUND: All available ARV (antiretroviral) agents can cause hepatotoxicity. Many case reports of ARV-induced hepatotoxicity have been described in patients with confounding viral hepatitis. This case series is comprised 23 HIV-positive patients with hepatic enzyme abnormalities but without the evidence of viral hepatitis. The data available for these 23 patients were assessed with an effort to establish any correlation between ARV therapy and abnormal liver function tests (LFTs) as well as the histologic findings on liver biopsies. METHODS: The 23 participants included in this study were referred to a hepatology/gastrointestinal clinic that catered specifically to HIV patients. The patients were referred by their HIV providers for evaluation of elevated LFTs, gastrointestinal symptoms or cirrhosis. The data surveyed included variables associated with hepatotoxicity and HIV infection. RESULTS: Liver biopsies were obtained in 21 out of 23 participants. The remaining two participants had evidence of cirrhosis based on imaging studies. The LFT elevations were definitely or possibly attributed to ARV therapy in 17 out of 23 participants. Specifically, the biochemical hepatotoxicity was definitely related to ARV therapy in six and possibly related to ARV medications in 11 participants. Nine out of 17 participants had evidence of nonalcoholic steatohepatitis, whereas four out of 17 had clinical features of lipodystrophy. Six participants had elevated LFTs before starting ARV therapy. The participants with nonalcoholic fatty liver diseases had normal LFTs for many years after which a steep rise was noted. All participants with nonalcoholic fatty liver diseases were exposed to nucleoside reverse transcriptase inhibitors. CONCLUSION: ARV medications, particularly the nucleoside reverse transcriptase inhibitors, can cause a dose-dependent hepatotoxicity that occurs after several months of exposure and possibly result in increasing the adverse effects of alcohol and obesity. Owing to the overlap of ARV medications, the contribution of each class of drugs toward the observed hepatotoxicity is not entirely clear. Liver biopsies should be considered in patients receiving ARV therapy with elevated LFTs and/or evidence of fatty liver.