Brian E. Schultz

Structural, biochemical, and biophysical characterization of idelalisib binding to phosphoinositide 3-kinase δ.

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 inhibitors interactions with PI3Kδ. Significance: Its use in humans makes it important to understand how idelalisib inhibits PI3Kδ. Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. A crystal structure of idelalisib bound to the p110δ subunit of PI3Kδ furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.

Role of Mitochondrial RNA Polymerase in the Toxicity of Nucleotide Inhibitors of Hepatitis C Virus

ABSTRACT Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4′-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2′-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2′-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing.

Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.

Discovery of Potent Cyclophilin Inhibitors Based on the Structural Simplification of Sanglifehrin A

Cyclophilin inhibition has been a target for the treatment of hepatitis C and other diseases, but the generation of potent, drug-like molecules through chemical synthesis has been challenging. In this study, a set of macrocyclic cyclophilin inhibitors was synthesized based on the core structure of the natural product sanglifehrin A. Initial compound optimization identified the valine-m-tyrosine-piperazic acid tripeptide (Val-m-Tyr-Pip) in the sanglifehrin core, stereocenters at C14 and C15, and the hydroxyl group of the m-tyrosine (m-Tyr) residue as key contributors to compound potency. Replacing the C18-C21 diene unit of sanglifehrin with a styryl group led to potent compounds that displayed a novel binding mode in which the styrene moiety engaged in a π-stacking interaction with Arg55 of cyclophilin A (Cyp A), and the m-Tyr residue was displaced into solvent. This observation allowed further simplifications of the scaffold to generate new lead compounds in the search for orally bioavailable cyclophilin inhibitors.

Binding kinetics, potency, and selectivity of the hepatitis C virus NS3 protease inhibitors GS-9256 and vedroprevir

BACKGROUND GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays. METHODS Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods. RESULTS GS-9256 and vedroprevir had measured Ki values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; Ki values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was >10,000-fold against all tested off-target proteases. Association rate constants of 4×10(5)M(-1)s(-1) and 1×10(6)M(-1)s(-1), respectively, were measured, and dissociation rate constants of 4.8×10(-5)s(-1) and 2.6×10(-4)s(-1) were determined. CONCLUSIONS GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics. GENERAL SIGNIFICANCE The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.

ASK1 contributes to fibrosis and dysfunction in models of kidney disease.

Oxidative stress is an underlying component of acute and chronic kidney disease. Apoptosis signal–regulating kinase 1 (ASK1) is a widely expressed redox-sensitive serine threonine kinase that activates p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase kinases, and induces apoptotic, inflammatory, and fibrotic signaling in settings of oxidative stress. We describe the discovery and characterization of a potent and selective small-molecule inhibitor of ASK1, GS-444217, and demonstrate the therapeutic potential of ASK1 inhibition to reduce kidney injury and fibrosis. Activation of the ASK1 pathway in glomerular and tubular compartments was confirmed in renal biopsies from patients with diabetic kidney disease (DKD) and was decreased by GS-444217 in several rodent models of kidney injury and fibrosis that collectively represented the hallmarks of DKD pathology. Treatment with GS-444217 reduced progressive inflammation and fibrosis in the kidney and halted glomerular filtration rate decline. Combination of GS-444217 with enalapril, an angiotensin-converting enzyme inhibitor, led to a greater reduction in proteinuria and regression of glomerulosclerosis. These results identify ASK1 as an important target for renal disease and support the clinical development of an ASK1 inhibitor for the treatment of DKD.

Direct Detection of Reactive Nitrogen Species in Experimental Autoimmune Uveitis

Purpose Demonstrate unequivocally the generation of nitric oxide in experimental autoimmune uveoretinitis by electron spin resonance spectroscopy (ESR) using ferrous iron complex of N-methyl-D-glucamine dithiocarbamate, (MGD)2-Fe2+, as a spin trap. Methods Experimental autoimmune uveitis was induced in Lewis rats, and at the peak of the intraocular inflammation, the animals received intravitreous injections of the spin trap. The retina and choroid dissected from the enucleated globes were subjected to ESR. Similarly, the retina and choroid obtained at the peak of experimental autoimmune uveo-retinitis (EAU) were placed in a vial containing luminal, and chemiluminescence was counted on a Packard liquid scintillation analyzer. Results The ESR three-line spectrum (g=2.04; aN=12.5 G) obtained was characteristic of the adduct [(MGD)2-Fe2+-NO]. The majority of this signal was eliminated by the inducible nitric oxide synthase (iNOS) specific inhibitor aminoguanidine injected inflamed retina was detected when compared with that of the non inflamed controls. The chemiluminescent activity was further increased two-fold by the addition of bicarbonate to the inflamed retina; the phenomenon is attributable only to the presence of a high steady-state concentration of peroxynitrite. Conclusions The study shows an unequivocal presence of nitric oxide in EAU retina and choroid and the generation of peroxynitrite. High levels of these reactive nitrogen species generated in the inflamed retina and choroids are certain to cause irreversible tissue damage, especially at the susceptible sites such as photoreceptors.

Rapid Formation of a Semiquinone Species on Oxidation of Quinol by the Cytochrome bo3 Oxidase from Escherichia coli

Many bacterial oxidases utilize dihydroquinols, such as ubiquinol or menaquinol, rather than cytochrome c as a substrate. The best-characterized ubiquinol oxidase is cytochrome bo 3 from Escherichia coli. In this work, the initial oxidation of ubiquinol by this ubiquinol oxidase is examined. Stopped-flow UV-visible spectroscopy and rapid freeze-quench electron paramagnetic resonance (EPR) spectroscopies were used to examine the oxidation of ubiquinol-2 (UQ2H2) by cytochrome bo 3 under multiple turnover conditions. The results show the rapid appearance of the semiquinone radical, coincident with the reduction of the low-spin heme b component of the enzyme. The rate of formation of the semiquinone radical is consistent with the proposition that this is a kinetically relevant intermediate in the reaction sequence. As UQ2H2 is depleted, the radical decays and the enzyme forms a “peroxy,” or P, complex with dioxygen. No detectable protein radical is associated with the P complex.

Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors

Influenza polymerase is a heterotrimer composed of polymerase acidic protein A (PA) and basic proteins 1 (PB1) and 2 (PB2). The endonuclease active site, located in the PA subunit, cleaves host mRNA to prime viral mRNA transcription, and is essential for viral replication. To date, the human influenza A endonuclease activity has only been studied on the truncated active-site containing N-terminal domain of PA (PAN) or full-length PA in the absence of PB1 or PB2. In this study, we characterized the endonuclease activity of recombinant proteins of influenza A/PR8 containing full length PA, PA/PB1 dimer, and PA/PB1/PB2 trimer, observing 8.3-, 265-, and 142-fold higher activity than PAN, respectively. Using the PA/PB1/PB2 trimer, we developed a robust endonuclease assay with a synthetic fluorogenic RNA substrate. The observed Km (150 ± 11 nM) and kcat [(1.4 ± 0.2) x 10-3s-1] values were consistent with previous reports using virion-derived replication complex. Two known influenza endonuclease phenylbutanoic acid inhibitors showed IC50 values of 10–20 nM, demonstrating the utility of this system for future high throughput screening.

Discovery of a Potent and Orally Bioavailable Cyclophilin Inhibitor Derived from the Sanglifehrin Macrocycle

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.