Tenofovir (TFV) is used in combination with other antiretroviral drugs for human immunodeficiency virus (HIV) treatment and prevention. TFV requires two phosphorylation steps to become pharmacologically active; however, the kinases that activate TFV in cells and tissues susceptible to HIV infection have yet to be identified. Peripheral blood mononuclear cells (PBMC), vaginal, and colorectal tissues were transfected with siRNA targeting nucleotide kinases, incubated with TFV, and TFV-monophosphate (TFV-MP) and TFV-diphosphate (TFV-DP) were measured using mass spectrometry–liquid chromatography. Adenylate kinase 2 (AK2) performed the first TFV phosphorylation step in PBMC, vaginal, and colorectal tissues. Interestingly, both pyruvate kinase isozymes, muscle (PKM) or liver and red blood cell (PKLR), were able to phosphorylate TFV-MP to TFV-DP in PBMC and vaginal tissue, while creatine kinase, muscle (CKM) catalyzed this conversion in colorectal tissue. In addition, next-generation sequencing of the Microbicide Trials Network MTN-001 clinical samples detected 71 previously unreported genetic variants in the genes encoding these kinases. In conclusion, our results demonstrate that TFV is activated in a compartment-specific manner. Further, genetic variants have been identified that could negatively impact TFV activation, thereby compromising TFV efficacy in HIV treatment and prevention.

Attempts to prevent HIV infection through pre-exposure prophylaxis (PrEP) include topical application of anti-HIV drugs to the mucosal sites of infection; however, a potential role for local drug metabolizing enzymes in modulating the exposure of the mucosal tissues to these drugs has yet to be explored. Here we present the first report that enzymes belonging to the cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) families of drug metabolizing enzymes are expressed and active in vaginal and colorectal tissue using biopsies collected from healthy volunteers. In doing so, we discovered that dapivirine and maraviroc, a non-nucleoside reverse transcriptase inhibitor and an entry inhibitor currently in development as microbicides for HIV PrEP, are differentially metabolized in colorectal tissue and vaginal tissue. Taken together, these data should help to guide the optimization of small molecules being developed for HIV PrEP.

Many clinically used antiviral drugs are nucleoside or nucleotide analogue drugs, which have a unique mechanism of action that requires intracellular phosphorylation. This dependence on intracellular activation presents novel challenges for the discovery and development of nucleoside/nucleotide analogue drugs. Contrary to many small molecule drug development programs that rely on plasma pharmacokinetics and systemic exposures, the precise mechanisms that result in efficacious intracellular nucleoside triphosphate concentrations must be understood in the process of nucleoside/nucleotide drug development. The importance is highlighted here, using the following as case studies: the herpes treatment acyclovir, the cytomegalovirus therapy ganciclovir, and human immunodeficiency virus (HIV) treatments based on tenofovir, which are also in use for HIV prophylaxis. For each drug, the specificity of metabolism that results in its activation in different cells or tissues is discussed, and the implications explored. Acyclovir's dependence on a viral enzyme for activation provides selective pressure for resistance mutations.Ganciclovir is also dependent on a viral enzyme for activation, and suicide gene therapy capitalizes on that for a novel oncology treatment. The tissue of most relevance for tenofovir activation depends on its use as treatment or as prophylaxis, and the pharmacogenomics and drug-drug interactions in those tissues must be considered. Finally, differential metabolism of different tenofovir prodrugs and its effects on toxicity risk are explored. Taken together, these examples highlight the importance of understanding tissue specific metabolism for optimal use of nucleoside/nucleotide drugs in the clinic.

We have developed a class of small-molecule cytotoxic compounds that are highly potent in an in vitro tubulin polymerization assay. Preclinical studies show that these drug candidates are potent against a range of cancer cell lines and may be well suited for the treatment of hematologic or solid tumors resistant to existing chemotherapy agents. We will present data on representative members of this class that inhibit cancer cell growth at nanomolar concentrations and are potent against a variety of cancer cell lines, including liver, breast, ovarian, and lung. The compounds induce cell cycle arrest in the G2M state within 24 hours with cell death occurring over multiple days. These tubulin inhibitors also behave as potent antiangiogenesis agents and inhibit endothelial tube formation in HUVEC cells at nanomolar concentrations. Functional assays using efflux pump inhibitors show that, compared to many major chemotherapy agents, our class of drug candidates is significantly less affected by the efflux pumps MDR1 and MRP1, which are commonly overexpressed as a mechanism of multidrug resistance. Our compounds show comparable potency against wild-type and cell lines developed to be resistant to other cancer drugs, while the potency of clinically relevant compounds such as paclitaxel or doxorubicin is reduced between 80x and about 3000x in the same assay. This feature suggests that our tubulin inhibitors may be active against tumors that are resistant to common cancer drugs. Overexpression of the β-III isotype of tubulin, which is known to occur in many aggressive and metastatic tumors, is another clinically relevant mechanism of resistance to microtubule-targeting anticancer agents. It has been correlated with significantly lower response to docetaxel-based chemotherapy in a number of cancers and is considered an indicator of resistance to paclitaxel and vinorelbine. We will present preclinical data showing that our compounds have similar activity against cells showing normal levels and those highly overexpressing β-III tubulin. The compounds display good in vitro physicochemical properties as well as favorable in vivo pharmacokinetics. In addition, we will present in vivo tolerability and efficacy data in mice. The ability of this class of tubulin inhibitors to maintain their efficacy across multiple drug-resistant cancer cell lines makes them attractive candidates for development as chemotherapy agents. In particular, a new anticancer agent that is less susceptible to major transporters and retains its potency when β-III tubulin is overexpressed could lead to more effective precision second-line therapy. Citation Format: Mohan Sivaraja, Sivan Sizikov, Nilantha Sirisoma, Tamari Kirtadze, Madhuri Chattopadhyay, Makena Ewald, Subhadra Dash, Anne Wong, Georg Neckermann, Elaine To, Stephanie Chang, Timothy P. Shiau, David C. Williams, Kevin M. Short, Angels Estiarte, Anirban Datta, David B. Kita. Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4851.