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.
We demonstrated a proof-of-principle concept of a label-free platform that enables nucleic acid sequencing by binding methodology. The system utilizes gold surfaces having high fidelity plasmonic nanohole arrays which are very sensitive to minute changes of local refractive indices. Our novel surface chemistry approach ensures accurate identification of correct bases at individual positions along a targeted DNA sequence on the gold surface. Binding of the correct base on the gold sensing surface triggers strong spectral variations within the nanohole optical response, which provides a high signal-to-noise ratio and accurate sequence data. Integrating our label-free sequencing platform with a lens-free imaging-based device, we reliably determined targeted DNA sequences by monitoring the changes within the plasmonic diffraction images. Consequently, this new label-free surface chemistry technique, integrated with plasmonic lens-free imaging platform, will enable monitoring multiple biomolecular binding events, which could initiate new avenues for high-throughput nucleic acid sequencing.
The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof-of-concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T-6PEG-TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA.
Summary BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF-mutant melanoma cell lines are more sensitive than wild-type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF-mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF-mutant melanomas, regardless of their sensitivity to BRAF inhibitors.
Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor (NtRTI) that is often administered as first-line therapy against human immunodeficiency virus type-1 (HIV-1) infection and acts as a chain terminator when incorporated into viral DNA. However, HIV-1 reverse transcriptase (RT) excises TFV in the presence of either ATP or pyrophosphate, which is an important drug resistance mechanism that would interfere with the effective treatment. Previous studies have shown conflicting results on excision efficiencies for TFV-terminated primer-templates derived from either primer binding site (PBS) or polypurine tract (PPT) sequences. To provide mechanistic insight into the variation in TFV removal from both sequences that are vital for the HIV-1 life cycle, we compared the efficiencies of removal reaction in response to sequence dependence via utilizing blocked PBS and PPT primer-templates. We found an enhanced TFV excision with PPT sequence over PBS sequence through ATP-mediated removal and a subsequent incorporation of ATP into the unblocked primers. Furthermore, the rate of pyrophosphorolytic excision of TFV from PPT sequence was 21-fold higher than that for the PBS sequence. However, the addition of efavirenz, nonnucleoside reverse transcriptase inhibitor (NNRTI), to the removal reaction effectively inhibits the TFV excision from both primers by forming a stable complex that would leave TFV inaccessible for excision. These results illuminate the degree of primer-template sequence contribution on TFV removal as well as increase our understanding of the molecular mechanism for the beneficial effects of widely used combinations of antiretroviral regimens in the context of synergistic antiviral activity and drug resistance.
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