In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by co-expressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy) phenyl) propanoic acid (OCF 3 Phe), 13 C/ 15 N-labeled p-methoxyphenylalanine (OMePhe), and 15 N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3 Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of E. coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in 1 H-15 N, 1 H-13 C HSQC and 19 F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3 Phe mutants of an active site tyrosine inhibited binding; incorporating 15 N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Background: Idelalisib is a PI3Kδ inhibitor used to treat hematological malignancies.Results: Idelalisib is selective, noncovalent, reversible, and ATP-competitive.Conclusion: The crystal structure helps explain the potency and selectivity of idelalisib. The biophysical and biochemical data clarify the details of the inhibitor's interactions with PI3Kδ.Significance: Its use in humans makes it important to understand how idelalisib inhibits PI3Kδ.

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As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC 50 ], 0.048 to 0.68 M). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC 50 , 1.5 M). The active 5=-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with K i /K m values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1=-CN and 2=-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability. The recent regulatory approval of two HCV nonstructural protein 3/4A (NS3/4A) protease inhibitors, telaprevir and boceprevir, has led to increased treatment response rates when given in combination with pegylated interferon (IFN) and ribavirin (RBV) for those with an HCV genotype 1 infection. However, these regimens are limited by the emergence of viral resistance, increased adverse events, inability to treat patients who are intolerant or contraindicated to IFN treatment, and decreased efficacy in many patient populations who are most in need of therapy, including those with advanced liver diseases and those infected with other HCV genotypes (4). Nucleotide inhibitors (NIs) have demonstrated great promise as direct-acting antivirals with broad genotype coverage, lack of preexisting variants with reduced susceptibility, a high barrier to resistance, and the ability to produce potent and durable antiviral responses (5-7). Following intrahepatic activation involving nucleotide kinases, the active 5=-triphosphates of NIs target the HCV nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase by serving as alternative substrates and nonobligate chain terminators of viral RNA synthesis. The initial HCV NIs to enter clinical trials were N-nucleosides, containing the natural C-N glycosidic linkage, with either 2=-C-Me or 4=-azido...

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.