HIV encodes an RNA directed DNA polymerase (reverse transcriptase, RT) that is an essential enzyme in the viral replication cycle. This enzyme catalyzes the synthesis of double stranded proviral DNA from single stranded genomic RNA via a bireactant-biproduct mechanism. The functional enzyme purified from virus particles is a complex consisting of two polypeptides of molecular weight 66,000 and 51,000. Two of the four classes of currently approved anti-HIV drugs, the nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleoside reverse transcriptase inhibitors (NNRTIs), act by inhibiting this enzyme. In this review each step of DNA synthesis catalyzed by the RT is described and the mechanism of inhibition of catalysis and termination of DNA synthesis by NRTIs is detailed. The individual steps in the catalytic cycle and the effects that the NRTIs have on them have been examined using transient kinetic analysis. The impact of stereoisomerism and resistance mutations on the rate of NRTI triphosphate incorporation (k(pol)), binding in the catalytic complex (K(d)) and the overall efficiency of incorporation (k(pol)/K(d)) are summarized for lamivudine, coviracil and zalcitabine. The results provide insight into the molecular forces and structural features that make these molecules effective inhibitors.
Respiratory syncytial virus (RSV) represents a threat to infants, the elderly, and the immunocompromised. RSV entry blockers are in clinical trials, but escape mutations challenge their potential. In search of RSV inhibitors, we have integrated a signature resistance mutation into are combinant RSV virus and applied the strain to high-throughput screening. Counter screening of candidates returned 14 confirmed hits with activity in the nano- to low-micromolar range. All blocked RSV polymerase activity in minigenome assays. Compound 1a (GRP-74915) was selected for development based on activity (EC50=0.21 µM, selectivity index (SI) 40), and scaffold. Resynthesis confirmed potency of the compound, which suppressed viral RNA synthesis in infected cells. However, metabolic testing revealed a short half-life in the presence of mouse hepatocyte fractions. Metabolite tracking and chemical elaboration combined with 3D-quantitative structure-activity relationship modeling yielded analogs (i.e. 8n: EC50=0.06 µM, SI 500) that establish a platform for the development of a therapeutic candidate.
Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCEThis study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale highthroughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for smallmolecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism ...
There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5′-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5′-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5′-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5′-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104. This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.
A Rh(I)-catalyzed carbocyclization reaction of alleneynones affords functionalized 2-alkylidene-3-vinylcyclohexenones and 2-alkylidene-3-vinylcyclopentenones. The scope, limitations, and utility of this triene-forming protocol have been examined and the results reported within.Selective and concise entry into molecular complexity via carbocyclization reactions of unsaturated carbon-carbon bonds is an important area in organometallic chemistry. 1 Recently, it was demonstrated that Rh(I)-catalyzed cycloisomerization reactions of allenes lead to trienyl-containing carbocycles, 2 heterocycles, 2 d-and e-lactams, 3 and d-lactones. 4 Combining these functional motifs with a cross-conjugated trienyl moiety provides functionally dense substructures and if chemical reactivity can be controlled, a means of gaining rapid access to molecular complexity.Using cross-conjugated trienes in complexity generating reactions requires novel approaches to controlling doublebond selectivity. 5 It was this control element that led us to consider the carbocyclization reactions of alkynones 6 because the resulting trienones would possess double bonds that are sterically biased and electronically differentiated by the carbonyl group. In this Letter we report on the assembly of the allene-ynones and their participation in Rh(I)-catalyzed carbocylization reactions to produce trienones. Selective reactions of the double bonds of the trienones were briefly investigated and also reported on.Allene-ynones 3a-j 7 were conveniently prepared by addition of the corresponding lithium acetylides 2a-j to amide 1 (Scheme 1). Compound 3k, possessing a terminal alkynone, was obtained by removal of the TMS group from 3e. 7c Subjecting allene-ynone 3e to the standard reaction protocol developed in our group for the Rh(I)-catalyzed cycloisomerization reaction {10 mol% [Rh(CO) 2 Cl] 2 , r.t.} afforded a 10% yield of 4e. Reasoning that alkynone 3e was either more reactive and/or trienone 4e less stable, the reaction was performed at 0°C using only 3 mol% of catalyst. 8 To our delight, trienone 4e was obtained in 95% yield after only five minutes at 0°C (entry 5, Table 1). Scheme 1 Formation of allenyl alkynones 3a-kNext, the scope and limitations of this reaction were examined by varying the substituents on the alkyne. These variations had a significant impact on the rate and yields of these cycloisomerization reactions. For example, if R 1 = Me (3a) or a TBS-protected propanol 3b, conversion to 4a or 4b required 90 minutes (entries 1 and 2, Table 1). Interestingly, shortening the tether between the protected alcohol and the alkyne from three to two methylene units dramatically reduces the reaction time to ca. 20 minutes (entries 3 and 4, Table 1). 9 Alternatively, placing an aromatic ring on the alkyne terminus slows the reaction, requiring that substrate 3g be heated to 50°C and 8 mol% of Rh(I) catalyst to effect the formation of trienone 4g. An electron-withdrawing or electron-donating group on the para-position of the aryl ring had a negligible effect on...
A series of 2'-substituted cyclobutyl nucleoside analogs were efficiently prepared by constructing the core cyclobutyl ring using different [2+2] cycloaddition approaches. The triphosphate derivative of a cyclobutyl nucleoside was also synthesized and evaluated against wild-type and mutant HIV reverse transcriptases (RT). Whereas the nucleoside analogs were inactive against HIV-1 in culture, the nucleotide showed good activity not only against wild-type and recombinant HIV RT (IC(50)=4.7, 6.9 microM), but also against the M184I and M184V mutants (IC(50)=6.1, 6.9 microM) in cell-free assays.
Forty-four tetracyclic hydroazulenoisoindoles were synthesized via a tandem cyclopropanation/ Cope rearrangement followed by a Diels-Alder sequence from easily available five-membered cyclic cross-conjugated trienones. These trienones were obtained from two different routes depending upon whether R 1 and R 2 are alkyl or amino acid derived functional groups, via a rhodium(I)-catalyzed cycloisomerization reaction. In order to increase diversity, four maleimides and two 1,2,4-triazoline-3,5-diones were used as dienophiles in the Diels-Alder step. Several Diels-Alder adducts were further reacted under palladium-catalyzed hydrogenation conditions, leading to a diastereoselective reduction of the trisubstituted double bond. This library has demonstrated rapid access to a variety of structurally complex natural product-like compounds via stereochemical diversity and building block diversity approaches.
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