Clarence R. Hurt

Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

Host–rabies virus protein–protein interactions as druggable antiviral targets

We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a host-catalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.

Capsid assembly as a point of intervention for novel anti-viral therapeutics.

In general, drug discovery in the therapeutic field of infectious disease has a stellar track record. And yet, subsets of pathogens, for example many classes of viruses other than HIV, HSV, influenza, and HCV, have been poorly addressed. In addition, the development of resistance remains a specter of great concern for almost all current chemotherapy directed against infectious diseases, including viruses. Within the viral lifecycle, capsid assembly stands out as a step occurring in all viruses, which has not been the subject of extensive drug discovery programs. Until recently, the common view of assembly was that all the necessary information for assembly was contained in the sequence of the viral protein, in other words, the capsid self-assembles. In the last few years, a body of data has opened new opportunities for antiviral pharmaceutical research. Evidence that host proteins may play catalytic or essential structural roles in viral capsid assembly suggests that these host proteins and their functions are novel targets for small molecule therapeutics. Here we review the current understanding of the capsid assembly process with emphasis on recent data that demonstrate the essential role of host proteins in capsid assembly. Furthermore, this dependency of assembly on host factors appears quite sensitive to small molecule intervention. Implications of this alternate mechanism of capsid assembly are also considered. For example, the dependency on host factors could impose a potent barrier to development of viral resistance to a host-targeted anti-capsid chemotherapeutic. Finally, we give specific examples of the current state of drug discovery programs that have focused on therapeutic inhibition of host-assisted viral capsid assembly.

Validation of a cell-based ELISA as a screening tool identifying anti-alphavirus small-molecule inhibitors.

Venezuelan (VEEV), eastern, and western equine encephalitis viruses, members of the genus Alphavirus, are causative agents of debilitative and sometimes fatal encephalitis. Although human cases are rare, these viruses pose a threat to military personnel, and to public health, due to their potential use as bioweapons. Currently, there are no licensed therapeutics for treating alphavirus infections. To address this need, small-molecules with potential anti-alphavirus activity, provided by collaborators, are tested routinely in live alphavirus assays utilizing time-consuming virus yield-reduction assays. To expedite the screening/hit-confirmation process, a cell-based enzyme-linked immunosorbent assay (ELISA) was developed and validated for the measurement of VEEV infection. A signal-to-background ratio of >900, and a z-factor of >0.8 indicated the robustness of this assay. For validation, the cell-based ELISA was compared directly to results from virus yield reduction assays in a single dose screen of 21 compounds. Using stringent criteria for anti-VEEV activity there was 90% agreement between the two assays (compounds displaying either antiviral activity, or no effect, in both assays). A concurrent compound-induced cell toxicity assay effectively filtered out false-positive hits. The cell-based ELISA also reproduced successfully compound dose-response virus inhibition data observed using the virus yield reduction assay. With available antibodies, this assay can be adapted readily to other viruses of interest to the biodefense community. Additionally, it is cost-effective, rapid, and amenable to automation and scale-up. Therefore, this assay could expedite greatly screening efforts and the identification of effective anti-alphavirus inhibitors.

Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals

BackgroundViral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus.ResultsHere, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures.ConclusionsThese results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner.ReviewersReviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers’ comments section.

The Emergence of Small-Molecule Inhibitors of Capsid Assembly as Potential Antiviral Therapeutics

Publisher Summary This chapter focuses on the emergence of small-molecule inhibitors of capsid assembly as potential antiviral therapeutics. There is a large degree of variation among viruses that cause human disease, but the general life cycle of viruses share common features that are potential targets for antiviral drugs. The features include viral attachment, viral entry, uncoating, replication, and release. Each of these key processes represents an opportunity to disrupt the viral life cycle and prevent the virus from replicating. Thus, in order for antiviral treatments to be effective, they must either block the entry of the virus into the cell or be active within the infected cell. Enzyme inhibitors have the unique ability to affect processes within infected cells. They bind to enzymes in a way that disrupts their function, preventing a step in the infectious process from occurring. The classes of enzymes currently targeted by antiviral therapy include reverse transcriptase, protease, integrase, replicase, and neuraminidase. Approaches that are employed to disrupt the viral life cycle include entry inhibitors, fusion inhibitors, and integrase inhibitors. The viral capsid is a structural protein that encloses and protects the genetic material of the virus during the viral replication process. Antivirals that interfere with the normal assembly or processing of the viral capsid are gaining momentum as potential therapies because the integrity of the viral capsid protein is crucial for infectivity and the replication of the virus.

Identification and Characterization of a Hepatitis C Virus Capsid Assembly Inhibitor

novel genotypic variant combinations was examined using chimeric sub-genomic replicons. Results: Population sequencing of the NS3 protease and NS5A regions in all baseline isolates revealed the pre-existence of one PI-resistant variant (1a-NS3-K155; ~20-fold increase in BMS650032 EC50). In five of six GT1a patients experiencing VBT, emergent resistance variants to BMS-650032 included substitutions at either NS3–155 (R155K) or NS3–168 (D168E/T/Y). Emergent resistance variants to BMS-790052 included substitutions at NS5A-30 (Q30R), NS5A-31 (L31V/M), and NS5A-93 (Y93C/N). In one patient demonstrating VBT, HCVRNA levels were too low (<100 IU/mL) for analysis. In the one patient who did not respond to the addition of pegIFNa/RBV, the NS3 protease and NS5A resistance variants persisted 4 weeks post-treatment. The patient with a preexisting PI-resistant variant demonstrated rapid viral load decline and remained undetectable throughout the treatment period but relapsed at WK4 post-treatment. Two GT1b patients responded to 2-DAA treatment and demonstrated no relapse 4 weeks posttreatment. Conclusions: HCV NS3 protease and NS5A variants reported to confer resistance to BMS-650032 and BMS-790052, respectively, were selected in prior null responders infected with HCV GT1a during the 2-DAA treatment. These variants were detected at the time of VBT. No VBT was observed in patients infected with GT1b or receiving two DAA with pegIFNa/RBV.