Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Insights into kinetic mechanism of Janus kinase 3 and its inhibition by tofacitinib.

JAK3 kinase plays a critical role in several cytokine signaling pathways involved in immune cell development and function. The studies presented in this report were undertaken to elucidate the kinetic mechanism of the JAK3 kinase domain, investigate the role of activation loop phosphorylation in regulating its catalytic activity, and examine its inhibition by the anti-rheumatoid arthritis drug, tofacitinib. Phosphorylation of two Tyr residues in JAK3's activation loop has been reported to impact its kinase activity. The recombinant JAK3 kinase domain used in our studies was heterogeneous in its activation loop phosphorylation, with the non-phosphorylated protein being the dominant species. Kinetic analysis revealed similar kinetic parameters for the heterogeneously phosphorylated JAK3, JAK3 mono-phosphorylated on Tyr 980, and the activation loop mutant YY980/981FF. Bisubstrate and product inhibition kinetic results were consistent with both sequential random and sequential ordered kinetic mechanisms. Solvent viscosometric experiments showed perturbation of kcat, suggesting the phosphoryl transfer step is not likely rate limiting. This was supported by results from quench-flow experiments, where a rapid burst of product formation was observed. Kinetic analysis of JAK3 inhibition by tofacitinib indicated inhibition is time dependent, characterized by on- and off-rate constants of 1.4 ± 0.1 µM-1s-1 and 0.0016 ± 0.0005 s-1, respectively.

Radioligand binding to intact cells as a tool for extended drug screening in a representative physiological context.

Radioligand binding assays on intact cells offer distinct advantages to those on membrane suspensions. Major pharmacological properties like drug affinity and binding kinetics are more physiologically relevant. Complex mechanisms can be studied with a wider choice of experimental approaches and so provide insights into induced-fit type binding, receptor internalisation and even into pharmacomicrokinetic phenomena like drug rebinding and partitioning into the membrane. Hence, intact cell binding constitutes a valuable addition to the pharmacologist's toolbox.

Contrasting roles of mitogen-activated protein kinases in cellular entry and replication of hepatitis C virus: MKNK1 facilitates cell entry.

The human kinome comprises over 800 individual kinases. These contribute in multiple ways to regulation of cellular metabolism and may have direct and indirect effects on virus replication. Kinases are tempting therapeutic targets for drug development, but achieving sufficient specificity is often a challenge for chemical inhibitors. While using inhibitors to assess whether c-Jun N-terminal (JNK) kinases regulate hepatitis C virus (HCV) replication, we encountered unexpected off-target effects that led us to discover a role for a mitogen-activated protein kinase (MAPK)-related kinase, MAPK interacting serine/threonine kinase 1 (MKNK1), in viral entry. Two JNK inhibitors, AS601245 and SP600125, as well as RNA interference (RNAi)-mediated knockdown of JNK1 and JNK2, enhanced replication of HCV replicon RNAs as well as infectious genome-length RNA transfected into Huh-7 cells. JNK knockdown also enhanced replication following infection with cell-free virus, suggesting that JNK actively restricts HCV replication. Despite this, AS601245 and SP600125 both inhibited viral entry. Screening of a panel of inhibitors targeting kinases that may be modulated by off-target effects of AS601245 and SP600125 led us to identify MKNK1 as a host factor involved in HCV entry. Chemical inhibition or siRNA knockdown of MKNK1 significantly impaired entry of genotype 1a HCV and HCV-pseudotyped lentiviral particles (HCVpp) in Huh-7 cells but had only minimal impact on viral RNA replication or cell proliferation and viability. We propose a model by which MKNK1 acts to facilitate viral entry downstream of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), both of which have been implicated in the entry process.

Transcriptomic analysis of the woodchuck model of chronic hepatitis B.

UNLABELLED: The Eastern woodchuck (Marmota monax) is naturally infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to the human hepatitis B virus (HBV). The woodchuck is used as an animal model for studying chronic hepatitis B (CHB) and HBV-associated hepatocellular carcinoma (HCC) in humans, but the lack of sequence information has hitherto precluded functional genomics analysis. To address this major limitation of the model, we report here the sequencing, assembly, and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. Using this new platform, we characterized the transcriptional response to persistent WHV infection and WHV-induced HCC. This revealed that chronic WHV infection, like HBV, is associated with (1) a limited intrahepatic type I interferon response; (2) intrahepatic induction of markers associated with T cell exhaustion; (3) elevated levels of suppressor of cytokine signaling 3 (SOCS3) in the liver; and (4) intrahepatic accumulation of neutrophils. Underscoring the translational value of the woodchuck model, this study also determined that WHV-induced HCC shares molecular characteristics with a subtype of human HCC with poor prognosis. CONCLUSION: Our data establish the translational value of the woodchuck model and provide new insight into immune pathways which may play a role either in the persistence of HBV infection or the sequelae of CHB.

Characterization of the elongation complex of dengue virus RNA polymerase: assembly, kinetics of nucleotide incorporation, and fidelity.

Dengue virus (DENV) infects 50-100 million people worldwide per year, causing severe public health problems. DENV RNA-dependent RNA polymerase, an attractive target for drug development, catalyzes de novo replication of the viral genome in three phases: initiation, transition, and elongation. The aim of this work was to characterize the mechanism of nucleotide addition catalyzed by the polymerase domain of DENV serotype 2 during elongation using transient kinetic methods. We measured the kinetics of formation of the elongation complex containing the polymerase and a double-stranded RNA by preincubation experiments. The elongation complex assembly is slow, following a one-step binding mechanism with an association rate of 0.0016 ± 0.0001 µM(-1) s(-1) and a dissociation rate of 0.00020 ± 0.00005 s(-1) at 37 °C. The elongation complex assembly is 6 times slower at 30 °C and requires Mg(2+) during preincubation. The assembled elongation complex incorporates a correct nucleotide, GTP, to the primer with a K(d) of 275 ± 52 µM and k(pol) of 18 ± 1 s(-1). The fidelity of the polymerase is 1/34,000, 1/59,000, 1/135,000 for misincorporation of UTP, ATP, and CTP opposite CMP in the template, respectively. The fidelity of DENV polymerase is comparable with HIV reverse transcriptase and the poliovirus polymerase. This work reports the first description of presteady-state kinetics and fidelity for an RNA-dependent RNA polymerase from the Flaviviridae family.

Why medicines work.

The interactions of candidate medicines with physiology that have yielded therapeutics are a small subset of the total interactions investigated. To be useful the interactions must initiate molecular actions that fix a disease phenotype. While much is known about the targets for successful interactions and the disease phenotypes, much less is understood of the molecular actions that connect the initial interactions to specific phenotypic changes. Towards a better understanding of these actions, the first in class drugs (233) approved between 1999 and 2020 by the United States FDA were analyzed. The analysis identifies the actions that have been successful and characteristics of those actions. The medicines clustered into a relatively few specific actions: those that act on systems through sensors/receptors and controllers (51%), those that act to disrupt essential functions (12%), and those that act to provide a molecular fix by repair, removal, or silencing (33%). Antimicrobials were clustered with those that disrupt essential functions and antivirals were clustered in the molecular category. The sensor and controller actions work through system specific regulatory nodes whereby a single modality triggers a change to a complex system. Actions that disrupt functions cause toxicity and death to cells and organisms, where in many cases, mechanisms of repair and compensation play a role in both death and specificity. The molecular actions directly address known disease causes and arise from the intersection between enabling technologies that identify disease cause, and development of new modalities and their corresponding actions that provide therapeutic solutions. Many of the medicines utilize physiologic processes involving committed transitions at the core of the actions to enhance specificity. These actions, which process the input to specific output, are important for understanding why medicines work.

3-Amino-pyrazolo[3,4-d]pyrimidines as p38α kinase inhibitors: design and development to a highly selective lead.

Learnings from previous Roche p38-selective inhibitors were applied to a new fragment hit, which was optimized to a potent, exquisitely selective preclinical lead with a good pharmacokinetic profile.

Steady-state kinetic characterization of kinase activity and requirements for Mg2+ of interleukin-1 receptor-associated kinase-4.

Interleukin-1 receptor-associated kinase-4 (IRAK-4) is a Ser/Thr-specific protein kinase that plays a critical role in intracellular signaling cascades mediated by Toll-like and interleukin-1 (IL-1) receptors. Despite a growing body of information on the physiological functions of IRAK-4, its kinase activity remains poorly studied. The present study entails characterization of the steady-state kinetic properties and Mg(2+) requirements of full-length, recombinant human IRAK-4 preactivated by incubation with MgATP. In the presence of 20 mM Mg(2+), activated IRAK-4 herein is demonstrated to phosphorylate a peptide substrate (IRAK-1 peptide), derived from the activation loop of IRAK-1, with a k(cat) of 30 +/- 2.9 s(-1) and K(m) values of 668 +/- 120 and 852 +/- 273 microM for ATP and the peptide, respectively. Two-substrate, dead-end and product inhibition data, using analogues of ATP, are consistent with both a sequential ordered kinetic mechanism with ATP binding to the enzyme prior to the peptide and a sequential random mechanism. Investigation of the Mg(2+) requirements for phosphoryl transfer activity of IRAK-4 revealed that more than one Mg(2+) ion interacts with the enzyme and that the enzyme is maximally active in the presence of 5-10 mM free Mg(2+). While one divalent metal, as part of a chelate complex with ATP, is essential for catalysis, kinetic evidence is provided to show that uncomplexed Mg(2+) further enhances the catalytic activity of IRAK-4 by bringing about an approximately 3-fold increase in k(cat) and an approximately 6-fold reduction in the K(m) for ATP and by rendering the interaction between the nucleotide and peptide substrate binding sites less antagonistic.

Fluxes for Unraveling Complex Binding Mechanisms.

A decade ago, many high-affinity drugs were thought to bind to their target via an induced-fit pathway instead of conformational selection. Yet, both pathways make up part of a thermodynamic cycle, and, owing to binding flux-based approaches, it is now rather considered that they act in parallel and also that their relative contribution to the final ligand-target complex depends on the ligand concentration. Those approaches are of increasing interest, but published data still merely refer to the peculiar situation of equilibrium binding. This article draws attention to the benefit of extending those approaches to address more physiological nonequilibrium binding conditions and in vivo situations. For the presented example, they help to apprehend transient experimental manifestations of a 'conventional' thermodynamic cycle.

Recent advances in phenotypic drug discovery.

There is a great need for innovative new medicines to treat unmet medical needs. The discovery and development of innovative new medicines is extremely difficult, costly, and inefficient. In the last decade, phenotypic drug discovery (PDD) was reintroduced as a strategy to provide first-in-class medicines. PDD uses empirical, target-agnostic lead generation to identify pharmacologically active molecules and novel therapeutics which work through unprecedented drug mechanisms. The economic and scientific value of PDD is exemplified through game-changing medicines for hepatitis C virus, spinal muscular atrophy, and cystic fibrosis. In this short review, recent advances are noted for the implementation and de-risking of PDD (for compound library selection, biomarker development, mechanism identification, and safety studies) and the potential for artificial intelligence. A significant barrier in the decision to implement PDD is balancing the potential impact of a novel mechanism of drug action with an under-defined scientific path forward, with the desire to provide infrastructure and metrics to optimize return on investment, which a known mechanism provides. A means to address this knowledge gap in the future is to empower precompetitive research utilizing the empirical concepts of PDD to identify new mechanisms and pharmacologically active compounds.

Development of a Bioluminescent High-Throughput Screening Assay for Nicotinamide Mononucleotide Adenylyltransferase (NMNAT).

Nicotinamide mononucleotide adenylyltransferase (NMNAT; EC 2.7.7.1) catalyzes the reversible production of NAD+ from NMN+ and ATP and is a potential drug target for cancer and neurodegenerative diseases. A sensitive bioluminescent assay format suitable to high-throughput screening (HTS) and mechanistic follow-up has not been reported and is of value to identify new modulators of NMNATs. To this end, we report the development of a bioluminescent assay using Photinus pyralis ATP-dependent luciferase and luciferin for NMNAT1 in a 384-well plate format. We also report a mechanistic follow-up paradigm using this format to determine time dependence and competition with substrates. The assay and follow-up paradigm were used to screen 912 compounds from the National Cancer Institute (NCI) Mechanistic Diversity Set II and the Approved Oncology Set VI against NMNAT1. Twenty inhibitors with greater than 35% inhibition at 20 µM were identified. The follow-up studies showed that seven actives were time-dependent inhibitors of NMNAT1. 2,3-Dibromo-1,4-naphthoquinone was the most potent, time-dependent inhibitor with IC50 values of 0.76 and 0.26 µM for inhibition of the forward and reverse reactions of the enzyme, respectively, and was shown to be NMN and ATP competitive. The bioluminescent NMNAT assay and mechanistic-follow-up will be of use to identify new modulators of NAD biosynthesis.

Biochemical mechanisms of drug action: what does it take for success?

Drug discovery is extremely difficult. There are many unanticipated scientific, medical and business challenges to every drug discovery programme. It is important to increase our understanding of the fundamental properties of effective drugs so that we can anticipate potential problems in developing new agents. This article addresses potential drug discovery and development risks associated with the biochemical mechanism of drug action, and proposes simple rules to minimize these risks.

Biochemical mechanisms of New Molecular Entities (NMEs) approved by United States FDA during 2001-2004: mechanisms leading to optimal efficacy and safety.

The United States FDA approved 85 New Molecular Entities (NMEs) during the period from January 2001 to November 2004 of which 60 were pharmaceuticals with known molecular targets. The majority targeted enzymes (48%) or G-protein coupled receptors (GPCRs) (33%). Eighty percent of the NMEs interacted at the same site as endogenous effector; either as competitive inhibitor/antagonist (67%) or agonist (13%). Three biochemical operations defined the modes of action of the NMEs: 1) mass action competition (equilibrium), 2) a drug stabilized conformational change in the target that is important to the response (conformational) and/or 3) drug action is less-responsive to mass action competition with effectors due to non-equilibrium kinetics (non-equilibrium kinetic). Approximately 80% of the NMEs elicit a response utilizing conformational and/or non-equilibrium kinetic mechanisms. The remaining 20% of NMEs find mass action competition with the endogenous substrate or ligand sufficient for therapeutic utility. These observations indicate that for the majority of drug targets, mass action driven equilibrium binding alone may not be sufficient for maximal therapeutic utility. A key determinant of the biochemical mode of action for these NMEs was to minimize the potential for toxicity, either by providing a maximal response at a low dose to minimize off-target toxicities, or by providing a mechanism to minimize the incidence of mechanism-based toxicity while retaining a sufficiently efficacious response. This principle appears to be independent of target class and provides insight as to intrinsic biochemical features and approaches required for a maximal therapeutic index.

Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates.

Protein kinases are an important class of enzymes and drug targets. New opportunities to discover medicines for neglected diseases can be leveraged by the extensive kinase tools and knowledge created in targeting human kinases. A valuable tool for kinase drug discovery is an enzyme assay that measures catalytic function. The functional assay can be used to identify inhibitors, estimate affinity, characterize molecular mechanisms of action (MMOAs) and evaluate selectivity. However, establishing an enzyme assay for a new kinases requires identification of a suitable substrate. Identification of a new kinase's endogenous physiologic substrate and function can be extremely costly and time consuming. Fortunately, most kinases are promiscuous and will catalyze the phosphotransfer from ATP to alternative substrates with differing degrees of catalytic efficiency. In this manuscript we review strategies and successes in the identification of alternative substrates for kinases from organisms responsible for many of the neglected tropical diseases (NTDs) towards the goal of informing strategies to identify substrates for new kinases. Approaches for establishing a functional kinase assay include measuring auto-activation and use of generic substrates and peptides. The most commonly used generic substrates are casein, myelin basic protein, and histone. Sequence homology modeling can provide insights into the potential substrates and the requirement for activation. Empirical approaches that can identify substrates include screening of lysates (which may also help identify native substrates) and use of peptide arrays. All of these approaches have been used with a varying degree of success to identify alternative substrates.

On the different experimental manifestations of two-state 'induced-fit' binding of drugs to their cellular targets.

'Induced-fit' binding of drugs to a target may lead to high affinity, selectivity and a long residence time, and this mechanism has been proposed to apply to many drugs with high clinical efficacy. It is a multistep process that initially involves the binding of a drug to its target to form a loose RL complex and a subsequent isomerization/conformational change to yield a tighter binding R'L state. Equations with the same mathematical form may also describe the binding of bivalent antibodies and related synthetic drugs. Based on a selected range of 'microscopic' rate constants and variables such as the ligand concentration and incubation time, we have simulated the experimental manifestations that may go along with induced-fit binding. Overall, they validate different experimental procedures that have been used over the years to identify such binding mechanisms. However, they also reveal that each of these manifestations only becomes perceptible at particular combinations of rate constants. The simulations also show that the durable nature of R'L and the propensity of R'L to be formed repeatedly before the ligand dissociates will increase the residence time. This review may help pharmacologists and medicinal chemists obtain preliminary indications for identifying an induced-fit mechanism.

Glycogen Synthase Kinase 3ß Promotes the Endocytosis of Transferrin in the African Trypanosome.

Human parasite Trypanosoma brucei proliferates in the blood of its host, where it takes up iron via receptor-mediated endocytosis of transferrin (Tf). Mechanisms of Tf endocytosis in the trypanosome are not fully understood. Small molecule lapatinib inhibits Tf endocytosis in T. brucei and associates with protein kinase GSK3ß (TbGSK3ß). Therefore, we hypothesized that Tf endocytosis may be regulated by TbGSK3ß, and we used three approaches (both genetic and small molecule) to test this possibility. First, the RNAi knock-down of TbGSK3ß reduced Tf endocytosis selectively, without affecting the uptake of haptaglobin-hemoglobin (Hp-Hb) or bovine serum albumin (BSA). Second, the overexpression of TbGSK3ß increased the Tf uptake. Third, small-molecule inhibitors of TbGSK3ß, TWS119 (IC50 = 600 nM), and GW8510 (IC50 = 8 nM) reduced Tf endocytosis. Furthermore, TWS119, but not GW8510, selectively blocked Tf uptake. Thus, TWS119 phenocopies the selective endocytosis effects of a TbGSK3ß knockdown. Two new inhibitors of TbGSK3ß, LY2784544 (IC50 = 0.6 µM) and sorafenib (IC50 = 1.7 µM), were discovered in a focused screen: at low micromolar concentrations, they prevented Tf endocytosis as well as trypanosome proliferation (GI50's were 1.0 and 3.1 µM, respectively). These studies show that (a) TbGSK3ß regulates Tf endocytosis, (b) TWS119 is a small-molecule tool for investigating the endocytosis of Tf,

A Four-Point Screening Method for Assessing Molecular Mechanism of Action (MMOA) Identifies Tideglusib as a Time-Dependent Inhibitor of Trypanosoma brucei GSK3ß.

BACKGROUND: New therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC50, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities. METHODOLOGY/PRINCIPAL FINDINGS: In this work we report a four point screening methodology to evaluate the time-dependence and competition for inhibition of GSK3ß protein kinase isolated from T. brucei. Using this method, we identified tideglusib as a time-dependent inhibitor whose mechanism of action is time-dependent, ATP competitive upon initial binding, which transitions to ATP non-competitive with time. The enzyme activity was not recovered following 100-fold dilution of the buffer consistent with an irreversible mechanism of action. This is in contrast to the T. brucei GSK3ß inhibitor GW8510, whose inhibition was competitive with ATP, not time-dependent at all measured time points and reversible in dilution experiments. The activity of tideglusib against T. brucei parasites was confirmed by inhibition of parasite proliferation (GI50 of 2.3 µM). CONCLUSIONS/SIGNIFICANCE: Altogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3ß inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3ß inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified.

Bi-substrate kinetic analysis of an E3-ligase-dependent ubiquitylation reaction.

Little is known about the kinetic mechanism of E3 ubiquitin ligases. This work describes basic methodology to investigate the kinetic mechanism of E3 ubiquitin ligases. The method used steady state, bi-substrate kinetic analysis of an E3 ligase-catalyzed monoubiquitylation reaction using ubiquitin-conjugated E2 (E2ub) and a mutant IkappaBalpha as substrates to evaluate whether the E3-catalyzed ubiquitin transfer from E2ub to protein substrate was sequential, meaning both substrates bound before products leaving, or ping pong, meaning that ubiquitin-conjugated E2 would bind, transfer ubiquitin to the E3, and debind before binding of protein substrate. The method requires the E3 reaction to be rate limiting and at steady state. This was accomplished through optimization of the conditions to ensure that the E3-dependent transfer of ubiquitin from E2ub to substrate was rate limiting. We observed a sequential bi-substrate E3-dependent ubiquitylation reaction on using E2UBCH7 and IkappaBalphaSS32/36EE (IkappaBalphaee as substrates and a partially purified Jurkat cell lysate as a source for the E3 ligase activity). The sequential bi-substrate kinetic mechanism is consistent with the formation of a ternary complex among E2UBCH7, IkappaBalphaSS32/36EE, and E3 before the transfer of ubiquitin from E2UBCH7 to IkappaBalphaSS32/36EE. The described method should be of use to characterize the kinetic mechanism of other E3 ligase-catalyzed ubiquitylation reactions.