SignificanceIndoleamine 2,3-dioxygenase (IDO1) is a heme protein that catalyzes the dioxygenation of tryptophan. Cells expressing this activity are able to profoundly alter their surrounding environment to suppress the immune response. Cancer cells exploit this pathway to avoid immune-mediated destruction. Through a range of kinetic, structural, and cellular assays, we show that two classes of highly selective inhibitors of IDO1 act by competing with heme binding to apo-IDO1. This shows that IDO1 is dynamically bound to its heme cofactor in what is likely a critical step in the regulation of this enzyme. These results have elucidated a previously undiscovered role for the ubiquitous heme cofactor in immune regulation, and it suggests that other heme proteins in biology may be similarly regulated.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC 50 of ϳ0.5 M, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.Hepatitis C virus (HCV), 1 a positive strand RNA virus of the Flaviviridae family, is the major etiological agent of post-transfusion and sporadic non-A, non-B hepatitis (1). An estimated 2-3% of the world population is chronically infected with HCV, which causes significant liver disease, cirrhosis, and can eventually lead to the development of hepatocellular carcinoma. In infected cells, translation of the viral RNA yields a 3011-residue polyprotein chain (2-4), which is subsequently cleaved to generate envelope and core proteins, for assembly of new virus particles and nonstructural enzymes essential for viral replication (5-7). Studies using recombinant NS5B polymerase have provided direct evidence for RNA-dependent RNA polymerase activity (8, 9), and this catalytic activity has been confirmed to be required for infectivity in chimpanzees (10).NS5B polymerase contains a hydrophobic C-terminal domain thought to be responsible for anchoring the protein to mammalian cell membranes. Removal of the C-terminal 21 residues has been reported to facilitate protein isolation from Escherichia coli without compromising RdRp activity (11). The HCV RdRp initiates RNA synthesis preferentially from the 3Ј terminus of the template RNA (12, 13-15) but lacks specificity for HCV RNA in vitro, because it readily utilizes heterologous nonviral templates (8). Based on crystallographic studies of the enzyme containing C-terminal truncations (16, 17), the hydrophobic tail present in the full-length ...

Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3 end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3 terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.Replication of plus-strand RNA viruses requires a multisubunit enzyme, the replicase, which is composed of viral and cellular factors (6). Biochemical characterization of eukaryotic replicases is limited because of difficulty in obtaining sufficient quantities of purified replicase. Furthermore, the hepatitis C virus (HCV) replicase has not been reported to accept exogenously provided RNAs. These results have prompted studies of the recombinant HCV RNA-dependent RNA polymerase (RdRp), the subunit responsible for phosphoryl transfer (9,16,17,26,31,38). While RdRps lack many properties of replicases, they are useful for characterizing some fundamental activities, such as the recognition of the initiation site and the kinetics of nucleotide polymerization (4,18,24).The HCV RdRp has recently been demonstrated to initiate RNA synthesis preferentially from the 3Ј terminus of the template RNA (16,17,26,31). Initiation from the 3Ј terminus raises a potential problem that viruses might encounter: cellular RNases that degrade even a few 3Ј nucleotides could prevent the initiation of viral RNA replication. Several mechanisms have been proposed that might allow RNA viruses to preserve or restore the sequences at the termini of their genome. These include base-pairing-dependent and base-pairing-independent recombination (12), priming by oligonucleotides aborted during the initiation of RNA synthesis (29), telomerase-like addition of a repeated sequence (33), and nontemplated nucleotide addition (7,12). Also, terminal adenylyl transferase activity was found to be associated with poliovirus polymerase 3D pol (30), possibly causing restoration of infectivity of poliovirus RNAs lacking the wild-type poly(A) tail.Recombinant HCV RdRp was reported to possess the ability to add nontemplated nucleotides to the 3Ј end of viral RNAs (5). However, this terminal transferase (TNTase) activity was later purported to be a cellular enzyme copurifying with the HCV RdRp...

Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the Flaviviridae family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEYH to DEYA) or both NTPase and helicase activity (motif I, GKT to GAT and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.The Flaviviridae family is comprised of three genera, Flavivirus (such as Yellow fever virus and Dengue virus types 1 to 4), Hepacivirus (such as Hepatitis C virus [HCV]), and Pestivirus (such as Bovine viral diarrhea virus [BVDV]) (28). BVDV infection represents an economically important disease of cattle, and BVDV has been identified as the causative agent of viral diarrhea-mucosal disease (reviewed in references 1, 12, 25, and 37). Like the other members of the Flaviviridae, BVDV is an enveloped, plus-stranded RNA virus whose genome consists of a nonsegmented single-stranded RNA molecule. BVDV genomic RNA is approximately 12.5 kb and encodes a single open reading frame of approximately 3,900 amino acids (7-9, 24). The polyprotein translated from the open reading frame is subsequently processed by virally encoded and cellular proteases into 12 individual proteins (13,30,31,35,44). These individual proteins function either as structural components of the virion or presumably, at least in part, as components of the viral RNA replicase complex as described for other Flaviviridae family members (2, 6, 17). RNA replicons derived from defective interfering particles have shown that the 5Ј and 3Ј nontranslated regions (NTRs) along with the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B can support RNA replication (4, 45). However, the essentiality of the individual nonstructural proteins has not yet been tested.The NS5B proteins of both BVDV (46) and the related HCV (3, 23) display RNA...

The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335-342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn(2+) concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.

Tumors can exploit the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to create an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan into immunosuppressive kynurenine, leading to suppressed effector T-cell (Teff) proliferation, allowing for tumor escape from host immune surveillance. IDO1 inhibition counteracts this immunosuppressive tumor microenvironment and may improve cancer outcomes, particularly when combined with other immunotherapies. Linrodostat mesylate (linrodostat) is a potent, selective oral IDO1 inhibitor that occupies the heme cofactor–binding site to prevent further IDO1 activation and is currently in multiple clinical trials for treatment of patients with advanced cancers. Here, we assess the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PKs) of linrodostat. Linrodostat exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 and HeLa cells stimulated with IFNγ, with no activity against tryptophan 2,3-dioxygenase or murine indoleamine 2,3-dioxygenase 2 detected. Linrodostat restored T-cell proliferation in a mixed-lymphocyte reaction of T cells and allogeneic IDO1-expressing dendritic cells. In vivo, linrodostat reduced kynurenine levels in human tumor xenograft models, exhibiting significant PD activity. Linrodostat demonstrated a PK/PD relationship in the xenograft model, preclinical species, and samples from patients with advanced cancers, with high oral bioavailability in preclinical species and low to moderate systemic clearance. Our data demonstrate that linrodostat potently and specifically inhibits IDO1 to block an immunosuppressive mechanism that could be responsible for tumor escape from host immune surveillance with favorable PK/PD characteristics that support clinical development.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing dioxygenase enzyme implicated in cancer immune response. This account details the discovery of BMS-986242, a novel IDO1 inhibitor designed for the treatment of a variety of cancers including metastatic melanoma and renal cell carcinoma. Given the substantial interest around this target for cancer immunotherapy, we sought to identify a structurally differentiated clinical candidate that performs comparably to linrodostat (BMS-986205) in terms of both in vitro potency and in vivo pharmacodynamic effect in a mouse xenograft model. On the basis of its preclinical profile, BMS-986242 was selected as a candidate for clinical development.

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