Current advancements in antiretroviral therapy (ART) have turned HIV-1 infection into a chronic and manageable disease. However, treatment is only effective until HIV-1 develops resistance against the administered drugs. The most recent antiretroviral drugs have become superior at delaying the evolution of acquired drug resistance. In this review, the viral fitness and its correlation to HIV-1 mutation rates and drug resistance are discussed while emphasizing the concept of lethal mutagenesis as an alternative therapy. The development of resistance to the different classes of approved drugs and the importance of monitoring antiretroviral drug resistance are also summarized briefly.

Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analogue prodrugs in addition to its natural substrates, 2′-deoxycytidine, 2′-deoxyguanosine, and 2′-deoxyadenosine. To improve the low catalytic activity and tailor the substrate specificity of dCK, we have constructed libraries of mutant enzymes and tested them for thymidine kinase (tk) activity. Random mutagenesis was employed to probe for amino acid positions with an impact on substrate specificity throughout the entire enzyme structure, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. Kinetic analysis indicates that Arg104Gln/Asp133Gly creates a "generalist" kinase with broader specificity and elevated turnover for natural and prodrug substrates. In contrast, the substitutions of Arg104Met/ Asp133Thr, obtained via site-saturation mutagenesis, yielded a mutant with reversed substrate specificity, elevating the specific constant for thymidine phosphorylation by over 1000-fold while eliminating activity for dC, dA, and dG under physiological conditions. The results illuminate the key contributions of these two amino acid positions to enzyme function by demonstrating their ability to moderate substrate specificity.Human deoxycytidine kinase (dCK; 1 EC 2.7.1.74) catalyzes the phosphorylation of 2′-deoxycytidine (dC), 2′-deoxyadenosine (dA) and 2′-deoxyguanosine (dG) to their corresponding monophosphates using nucleoside triphosphates as phosphoryl donors. This reaction is the first step of the deoxyribonucleoside salvage pathway, an alternative to de novo nucleotide biosynthesis, which, in combination with deoxyribonucleoside mono-and diphosphate kinases, provides triphosphate anabolites for DNA replication and repair (1).In addition to recycling natural 2′-deoxyribonucleosides, dCK catalyzes the initial, often ratedetermining phosphorylation of several chemotherapeutic nucleoside analogue (NA) prodrugs such as gemcitabine (2′,2′-difluorodeoxycytidine), AraC (1-β-D-arabinosylcytosine), and clofarabine [2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-9H-purin-6-amine], as well as antiviral prodrugs including ddC (2′,3′-dideoxycytidine), 3TC (2′-deoxy-3′-thiacytidine), and FTC (5-fluoro-2′-deoxy-3′-thiacytidine) whose pharmacological activity depends on their triphosphate form (2-7). Given the critical role of dCK in phosphorylating 2′- † The authors would like to acknowledge financial support in part by NIH Grant GM69958 and by a grant to the Emory Center for AIDS Research (AI050409) from the NIH and by institutional funding from the Emory University Health Science Center. *Corresponding author: Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322. Tel: (404) 712-2170. Fax: (404) 727-6586. E-mail: sal2@emory.edu. SUPPORTING INFORMATION AVAILABLE Oligonucleotide sequences used as primers for the site-directed mutagenesis (Table S-1) and statistical data on the codon distribution of the site-saturation mutage...

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a major target for currently approved anti-HIV drugs. These drugs are divided into two classes: nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the synthesis and biochemical evaluation of a novel bifunctional RT inhibitor utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI) linked via a poly(ethylene glycol) (PEG) linker. HIV-1 RT successfully incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in a base-specific manner. Moreover, this inhibitor demonstrates low nanomolar potency that has 4.3-fold and 4300-fold enhancement of polymerization inhibition in vitro relative to the parent TMC-derivative and d4T, respectively. This study serves as a proof-of-concept for the development and optimization of bifunctional RT inhibitors as potent inhibitors of HIV-1 viral replication.

Targeting anti-apoptotic proteins can sensitize tumor cells to conventional chemotherapies or other targeted agents. Antagonizing the Inhibitor of Apoptosis Proteins (IAPs) with mimetics of the pro-apoptotic protein SMAC is one such approach. We used sensitization compound screening to uncover possible agents with the potential to further sensitize lung adenocarcinoma cells to the SMAC mimetic Debio 1143. Several compounds in combination with Debio 1143, including taxanes, topoisomerase inhibitors, and bromodomain inhibitors, super-additively inhibited growth and clonogenicity of lung adenocarcinoma cells. Co-treatment with Debio 1143 and the bromodomain inhibitor JQ1 suppresses the expression of c-IAP1, c-IAP2, and XIAP. Non-canonical NF-κB signaling is also activated following Debio 1143 treatment, and Debio 1143 induces the formation of the ripoptosome in Debio 1143-sensitive cell lines. Sensitivity to Debio 1143 and JQ1 co-treatment was associated with baseline caspase-8 expression. In vivo treatment of lung adenocarcinoma xenografts with Debio 1143 in combination with JQ1 or docetaxel reduced tumor volume more than either single agent alone. As Debio 1143-containing combinations effectively inhibited both in vitro and in vivo growth of lung adenocarcinoma cells, these data provide a rationale for Debio 1143 combinations currently being evaluated in ongoing clinical trials and suggest potential utility of other combinations identified here.