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Inverse agonists of the nuclear receptor RORC2 have been widely pursued as a potential treatment for a variety of autoimmune diseases. We have discovered a novel series of isoindoline-based inverse agonists of the nuclear receptor RORC2, derived from our recently disclosed RORC2 inverse agonist 2. Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (20), which combined potent inhibition of IL-17A secretion from primary human TH17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical in vivo studies, compound 20 reduced thymocyte numbers in mice and showed dose-dependent reduction of IL-17A containing γδ-T cells and of IL-17A and IL-22 RNA in the imiquimod induced inflammation model. Based on these data and a favorable safety profile, 20 was progressed to phase 1 clinical studies.

RORγt, an isoform of the retinoic acid-related orphan receptor gamma (RORc, RORγ), has been identified as the master regulator of T-helper 17 (TH 17) cell function and development, making it an attractive target for the treatment of autoimmune diseases. Validation for this target comes from antibodies targeting interleukin-17 (IL-17), the signature cytokine produced by TH 17 cells, which have shown impressive results in clinical trials. Through focused screening of our compound collection, we identified a series of N-sulfonylated benzoxazepines, which displayed micromolar affinity for the RORγ ligand-binding domain (LBD) in a radioligand binding assay. Optimization of these initial hits resulted in potent binders, which dose-dependently decreased the ability of the RORγ-LBD to interact with a peptide derived from steroid receptor coactivator 1, and inhibited the release of IL-17 secretion from isolated and cultured human TH 17 cells with nanomolar potency. A cocrystal structure of inverse agonist 15 (2-chloro-6-fluoro-N-(4-{[3-(trifluoromethyl)phenyl]sulfonyl}-2,3,4,5-tetrahydro-1,4-benzoxazepin-7-yl)benzamide) bound to the RORγ-LBD illustrated that both hydrophobic interactions, leading to an induced fit around the substituted benzamide moiety of 15, as well as a hydrogen bond from the amide NH to His479 seemed to be important for the mechanism of action. This structure is compared with the structure of agonist 25 (N-(2-fluorophenyl)-4-[(4-fluorophenyl)sulfonyl]-2,3,4,5-tetrahydro-1,4-benzoxazepin-6-amine ) and structures of other known RORγ modulators.

The development of a scalable chiral synthesis for the HCV NS5B inhibitor MK-3281 is being reported. Several alternative routes were explored and are being described.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

A series of 2-(3-thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acid inhibitors of the hepatitis C virus (HCV) NS5B polymerase enzyme are reported. Sulfonyl urea substituted analogs in this series proved to be the most potent active site non-nucleoside inhibitors of NS5B reported to date. These compounds had low nanomolar enzyme inhibition across HCV genotypes 1-3 and showed single digit micromolar inhibition in the HCV replicon assay. This improved cell-based activity allowed the binding mode of these compounds to be probed by selection of resistant mutations against compound 21. The results generated are in broad agreement with the previously proposed binding model for this compound class.

Infections caused by the hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The NS5B polymerase of HCV is responsible for the replication of viral RNA and has been a prime target in the search for novel treatment options. We had discovered allosteric finger-loop inhibitors based on a thieno[3,2-b]pyrrole scaffold as an alternative to the related indole inhibitors. Optimization of the thienopyrrole series led to several N-acetamides with submicromolar potency in the cell-based replicon assay, but they lacked oral bioavailability in rats. By linking the N4-position to the ortho-position of the C5-aryl group, we were able to identify the tetracyclic thienopyrrole 40, which displayed a favorable pharmacokinetic profile in rats and dogs and is equipotent with recently disclosed finger-loop inhibitors based on an indole scaffold.

The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.

A variety of new prodrugs of 2'-methyl cytidine based on acyloxy ethylamino phosphoramidates have been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent drug a 10- to 20-fold increase in formation of nucleotide triphosphate in rat and human hepatocytes could be achieved.

The RNA replication machinery of HCV is a multi-subunit membrane–associated complex. NS5A has emerged as an active component of HCV replicase, possibly involved in regulation of viral replication and resistance to the antiviral effect of interferon. We report here substituted piperazinyl- N -(aryl)benzamides as potent inhibitors of HCV replication exerted via modulation of the dimerization of NS5A.

The global prevalence of hepatitis C virus (HCV) infection and the serious consequences associated with the chronic state of the disease have become a worldwide health problem. A combination therapy comprising Interferon-alpha and Ribavirin represents the current standard treatment for chronic HCV infection, although it has demonstrated limited success and causes serious side effects. Promising alternative approaches toward the control of HCV infection include the development of small molecule inhibitors of viral enzymes interfering with the essential steps in the life cycle of the virus. In this review we will focus on inhibitors of the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) which is essential for viral replication and has been recognized as a prime target for therapeutic intervention.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral RNA. We recently disclosed dihydroxypyrimidine carboxylates 2 as novel, reversible inhibitors of the HCV NS5B polymerase. This series was further developed into 5,6-dihydroxy-2-(2-thienyl)pyrimidine-4-carboxylic acids such as 34 (EC50 9.3 microM), which now show activity in the cell-based HCV replication assay. The structure-activity relationship of these inhibitors is discussed in the context of their physicochemical properties and of the polymerase crystal structure. We also report the results of mutagenesis experiments which support the proposed binding model, which involves pyrophosphate-like chelation of the active site Mg ions.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC(50) = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.

Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here a novel class of allosteric inhibitor of NS5B that shows potent affinity for the NS5B enzyme and effective inhibition of subgenomic HCV RNA replication in HUH-7 cells. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTPbinding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase. Hepatitis C virus (HCV) is the causative agent of the majority of chronic liver disease throughout the world. More than 170 million individuals are estimated to be infected with this virus (27). The size of the HCV epidemic and the limited efficacy of current therapy (based on the use of alpha interferon) have stimulated intense research efforts towards the development of antiviral drugs that are both better tolerated and more effective. The most widely established strategy for developing novel anti-HCV therapeutics aims at the identification of low-molecular-weight inhibitors of essential HCV enzymes.

Hepatitis C virus (HCV) infection is an epidemic disease and a significant worldwide health problem. Despite impressive improvements in the efficacy of the standard, interferon-based therapies, at present, the virus can not be eradicated in the majority of infected individuals. The last decade has witnessed a burst in our understanding of the molecular biology of HCV infection and lead to the identification of essential features of the viral genome that are being targeted for the development of specific antiviral agents. The non-structural protein 3 of the HCV genome harbours a serine protease domain that is essential for viral replication. This enzyme has been studied in great detail and the wealth of structural and functional data are presently nurturing drug development efforts. The peculiar active site structure of the enzyme imposes considerable obstacles to the development of small molecule inhibitors. However, the combination of creativity with the powerful tools of modern drug discovery has led to impressive progress in this field over the past few years and, as a result, the first compounds are now entering clinical trials.

In an industry focused on reducing attrition, lead generation represents a critical stage in drug discovery wherein a considered choice of lead series can significantly reduce the time and resources committed to unpromising chemical series. To aid identification and selection of series with a specific mechanism of action, within property space suitable for the intended mode of administration, many modalities are brought into play and are discussed in this chapter. Aspects covered include the approaches used to find chemical start points, as well as some of the blind alleys that the investigator should avoid, and the need to consider lead-like properties in context of a line-of-sight to the final drug candidate. By way of help, examples are given of both successful and unsuccessful lead generation.

The difluoromethyl group was designed by computational chemistry methods as a mimetic of the canonical P1 cysteine thiol for inhibitors of the hepatitis C virus NS3 protease. This modification led to the development of competitive, non-covalent inhibitor 4 (Ki 30 nM) and reversible covalent inhibitors (6, Ki 0.5 nM; and 8 Ki∗ 10 pM).

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.

Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N–terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His–Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His–Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes.

The hepatitis C virus NS3 protein contains a serine protease domain with a chymotrypsin-like fold, which is a target for development of therapeutics. We report the crystal structures of this domain complexed with NS4A cofactor and with two potent, reversible covalent inhibitors spanning the P1–P4 residues. Both inhibitors bind in an extended backbone conformation, forming an anti-parallel β-sheet with one enzyme β-strand. The P1 residue contributes most to the binding energy, whereas P2–P4 side chains are partially solvent exposed. The structures do not show notable rearrangements of the active site upon inhibitor binding. These results are significant for the development of antivirals.

Hepatitis C virus (HCV) is the major causative agent of non-A non-B hepatitis, an important health problem with an estimated 50 million people infected worldwide. Among the possible targets for therapeutic intervention, the serine protease contained within the N-terminal region of nonstructural protein 3 (NS3 protease) is so far the best characterized. In vitro characterization of synthetic substrates based on all the natural cleavage sites (as well as a series of analogs) has consistently revealed poor kinetic parameters, making them unsuitable for sensitive high-throughput screening. To overcome these difficulties, we have recently developed depsipeptide substrates incorporating an ester bond between residues P1 and Pprime1. Due to ready transesterification of the scissile bond to the acyl-enzyme intermediate, these substrates showed very high kcat/Km values, enabling detection of activity with subnanomolar NS3 concentrations. We have used the same principle to synthesize internally quenched depsipeptide fluorogenic substrates based on resonance energy transfer between the donor/acceptor couple 5-[(2'-aminoethyl)amino]naphthalene sulfonic acid/4-[[4'-(dimethylamino)phenyl]azo]benzoic acid, and developed a continuous assay for NS3 activity. Substrate cleavage is linear with enzyme concentration: depending on the conditions chosen, we estimated a detection limit for NS3 between 1 nM and 250 pM. The suitability of the assay for evaluation of inhibitors was established using as competitor a tridecapeptide corresponding to the natural NS4A/4B cleavage site; this gave an IC50 of 30 microM, well in agreement with the previously found Km value (40 microM).

We report a new series of inhibitors for hepatitis C virus NS5B RNA polymerase containing a constrained pentacyclic scaffold. Our SAR studies led to the identification of hexahydroindolo[2,1-a]pyrrolo[3,2-d][2]benzazepines exposing basic groups. The compounds displayed a high activity in the enzyme assay and displayed good activity in the cell-based (replicon) assay in the presence of serum proteins.

We report a new series of inhibitors for hepatitis C virus NS5B RNA polymerase containing a constrained pentacyclic scaffold. Our SAR studies led to the identification of hexahydroindolo[2,1-a]pyrrolo[3,2-d][2]benzazepines exposing basic groups. The compounds displayed a high activity in the enzyme assay and displayed good activity in the cell-based (replicon) assay in the presence of serum proteins.

Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here 2 series of indole-N-acetamides, bearing physicochemically diverse carboxylic acid replacements, which show potent affinity for the NS5B enzyme with reduced potential for formation of glucuronide conjugates. Preliminary optimization of these series furnished compounds that are potent in the blockade of subgenomic HCV RNA replication in HUH-7 cells.

The human and monetary costs of chronic hepatitis C and the complications arising from this disease emphasize the urgency to find a treatment for Hepatitis C Virus (HCV) infected patients. The current standard of treatment for patients chronically infected with HCV is combination therapy with pegylated interferon plus ribavirin. Recently, viral enzymes have become the target of efforts to develop small molecule inhibitors interfering with the essential steps in the life cycle of the virus. Amongst these enzymes the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) is essential for viral replication and has been recognized as a prime target for therapeutic intervention. Several distinct classes of inhibitors of NS5B RdRp have been disclosed in the literature, including active site inhibitors such as nucleosides and pyrophosphate mimetics, as well as non-nucleoside inhibitors. The latter, based on the success of allosteric inhibitors in the treatment of HIV infection, have been developed into compounds which show activity in the subgenomic cell-culture assay of HCV replication. This review provides an account of the recent developments in this field.

Thieno[3,2-b]pyrroles are a novel class of allosteric inhibitors of HCV NS5B RNA-dependent RNA polymerase which show potent affinity for the NS5B enzyme. Introduction of a polar substituent in the position N1 led to a compound that efficiently blocks subgenomic HCV RNA replication in HUH-7 cells with an EC50 of 2.9 μM.

5,6-Dihydroxypyrimidine-4-carboxylic acids are a promising series of hepatitis C virus (HCV) NS5B polymerase inhibitors that bind at the active site of the enzyme. Here we report a simple 2-thienyl substituted analogue that shows 10-fold improved activity over the original lead, and which allowed us to further delineate the key elements of the pharmacophore of this class of inhibitor. This work led to the identification of a trifluoromethyl acylsulfonamide group as a viable replacement for the C4 carboxylic acid in this series.

SAR on the phenethylamide 1 (Ki 1.2 μM) in the P2- and the P′-position led to potent inhibitors, one of which showed good exposure and low clearance when administered intramuscularly to rat.

The N-terminal aminoacid of phenethylamide tripeptide inhibitors of the hepatitis C virus NS3 protease can be replaced with an alpha-hydroxy acid to obtain more 'drug like' inhibitors with low micromolar activity. The preferred S-configuration of the capping residue can be explained by molecular modeling studies.

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

N-terminal truncation of the hexapeptide ketoacid 1 gave rise to potent tripeptide inhibitors of the hepatitis C virus NS3 protease/NS4A cofactor complex. Optimization of these tripeptides led to ketoacid 30 with an IC50 of 0.38 microM. The SAR of these tripeptides is discussed in the light of the recently published crystal structures of a ternary tripetide/NS3/NS4A complexes.

Hepatitis C is a predominantly chronic viral infection, affecting 1-3% of the world population. The causative agent, the hepatitis C virus (HCV), has a positive strand-RNA genome that is utilized, in infected cells, as an mRNA to drive the synthesis of a large polyprotein precursor. This precursor subsequently undergoes proteolytic maturation to generate all of the functional, both structural and nonstructural proteins necessary for viral replication and assembly. The proteolytic activity that is responsible for the generation of the mature viral polymerase as well as for most of the cleavages occurring in the nonstructural region of the polyprotein is expressed by the virus itself and is contained in its nonstructural protein 3 (NS3). Here, the N-terminal 180 amino acids form a chymotrypsin-like serine protease domain. Full activation of this protease is achieved only after complexation with another viral protein, the cofactor protein NS4A. Together, NS3 and NS4A form the active, heterodimeric serine protease that presently is the target of medicinal chemistry efforts aiming at the development of inhibitors with potential antiviral activity. We here review the recent progress in our understanding of the structure and function of the enzyme and in the development of selective and potent NS3 protease inhibitors.

The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the five cleavage events that occur in the nonstructural region of the HCV polyprotein. We here show that hexapeptide, tetrapeptide, and tripeptide alpha-ketoacids are potent, slow binding inhibitors of this enzyme. Their mechanism of inhibition involves the rapid formation of a noncovalent collision complex in a diffusion-limited, electrostatically driven association reaction followed by a slow isomerization step resulting in a very tight complex. pH dependence experiments point to the protonated catalytic His 57 as an important determinant for formation of the collision complex. K(i) values of the collision complexes vary between 3 nM and 18.5 microM and largely depend on contacts made by the peptide moiety of the inhibitors. Site-directed mutagenesis indicates that Lys 136 selectively participates in stabilization of the tight complex but not of the collision complex. A significant solvent isotope effect on the isomerization rate constant is suggestive of a chemical step being rate limiting for tight complex formation. The potency of these compounds is dominated by their slow dissociation rate constants, leading to complex half-lives of 11-48 h and overall K(i) values between 10 pM and 67 nM. The rate constants describing the formation and the dissociation of the tight complex are relatively independent of the peptide moiety and appear to predominantly reflect the intrinsic chemical reactivity of the ketoacid function.

The substrate specificity of a purified protein encompassing the hepatitis C virus NS3 serine protease domain was investigated by introducing systematic modifications, including non-natural amino acids, into substrate peptides derived from the NS4A/NS4B cleavage site. Kinetic parameters were determined in the absence and presence of a peptide mimicking the protease co-factor NS4A (Pep4A). Based on this study we draw the following conclusions: (i) the NS3 protease domain has an absolute requirement for a small residue in the P1 position of substrates, thereby confirming previous modelling predictions. (ii) Optimization of the P1 binding site occupancy primarily influences transition state binding, whereas the occupancy of distal binding sites is a determinant for both ground state and transition state binding. (iii) Optimized contacts at distal binding sites may contribute synergistically to cleavage efficiency.