ML322, A Small Molecule Inhibitor of Dimerization of the Core Protein of Hepatitis C Virus (HCV)

Over three million Americans are infected by the Hepatits C Virus (HCV) virus 2 which is particularly lethal for Acquired Immunodeficiency Syndrome (AIDS) patients, of whom increasing numbers are co-infected with Human Immunodeficiency Virus infection (HIV) and HCV. There is no vaccine, and the only available treatment, based on a combination of interferon and ribavirin, cures less than half of the patients. One of the important structural proteins of HCV is the core protein. HCV core has many reported functions in the host cells, but its main purpose for the virus is to enclose and thereby protect the HCV Ribonucleic acid (RNA) genome during disease transmission. The conserved nature of HCV and absence of a vaccine, along with studies demonstrating that core contributes to host cell oncogenesis, apoptosis inhibition, and suppression of host T cell responses, support a role for core as a major pathogenic component of HCV. As a result, the identification of specific inhibitors of HCV core dimerization may provide tools for inhibiting HCV assembly without host cell effects. Several studies identify HCV capsid assembly as an appropriate target for chemotherapeutic intervention of HCV infections. Thus, this High Throughput Screen (HTS) program was initiated to identify small molecules that inhibit HCV core protein dimerization. Following an HTS effort and several rounds of medicinal chemistry, we have identified compound CID 49800087 as inhibitor probe (ML322) belonging to the [1,2,4]triazolo[1,5-]pyrimidin-2-yl scaffold. The probe inhibits dimerization of core proteins in biochemical Alphascreen assays (IC50 = 8.4 μM; 51% inhibition at 15 μM). Using quantitative polymerase chain reactions (QPCR) the probe was shown to reduce levels of total HCV RNA in infected cells (IC50 = 1.8 μM) and significantly reduces levels of infectious HCV RNA in cells treated with HCV-containing culture supernatants (IC50 = 2 nM). Profiling revealed that probe ML322 lacks activity against a panel of 63 receptors, transporters, and ion channels (Ricerca). Modest activity was detected at muscarinic M1-3, adrenergic alpha, and dopamine transporter (DAT). Critically, probe ML322 is not cytotoxic against the Huh7.5 liver cell line used for the infectivity assays (CC50 = 33 μM). This novel probe represents an exciting tool for use in basic HCV research to elucidate the mechanisms by which core-core dimerization contributes to HCV nucleocapsid formation, HCV infection, and liver cancers associated with HCV.