Significance:

Despite breakthroughs in the treatment of viral diseases, they remain enormous public health challenges. Over 30 million people are currently infected with Human Immunodeficiency Virus (HIV). Remarkably, despite the availability of a vaccine for Hepatitis B Virus (HBV), ~400 million individuals are chronically infected and in need of treatment with antivirals. In this era of globalization, worldwide outbreaks of new, emerging, and re-emerging diseases including Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS), or Foot-and-Mouth-Disease (FMD) are global threats that have the potential to become an immense burden on the US and world economies. Recently, we have been living a pandemic, COVID-19, that is caused by coronavirus SARS-CoV-2, and that has resulted in millions of deaths throughout the globe. Our laboratory is working hard on contributing to the understanding of coronavirus biology, developing tools and helping develop therapeutics against SARS-CoV-2.

Academic research is the workhorse of drug discovery, as it focuses on fundamental virus biology and molecular interactions that involve viral targets, potential therapeutics, drug resistant proteins, and host factors. In depth understanding of these interactions is needed for innovative and novel drug discoveries and this is an area where we have made significant contributions. Toward that end we use a combination of crystallographic, biochemical, virological approaches to chart the details of molecular recognition during steps of the life cycle of viruses, allowing us to identify and target weak spots.

Research Projects

Project Title: Ultrapotent Inhibitors of Wild-type and Multi-drug Resistant HIV

Project PI: Sarafianos, Stefan G (Contact PI)

Project Number: R37AI076119

Project Description: Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) efficiently suppress HIV and serve as backbone of antiretroviral therapies. However, new therapeutics are needed for continued control of HIV infection. 4’- Ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) is an NRTI with exceptional potency, stability, and unique mechanism of action against HIV, which has been licensed by Merck. EFdA (also known as MK-8591 or Islatavir) has been successfully used in Phase I and recently introduced in Phase II clinical trials. EFdA has generated “compelling early results for both treatment and prevention” in patients, tested at remarkably low doses (up to >10,000-fold lower doses than current NRTI drugs) aiming at once-weekly oral and once-yearly slow-release dosing, unprecedented modalities for HIV therapies. Hence, this work is directly relevant to NIH guidelines for high priority AIDS funding (NOT-OD-15-137) for “next generation therapies…that are longer acting.” We have shown that a) EFdA has high potency in vitro, in cell culture (EC50=50 pM in PBMCs), mice and non- human primate animal models. Although EFdA retains the 3’-OH, it blocks HIV reverse transcriptase (RT) in vitro, primarily as an immediate/obligate and at times delayed chain terminator due to difficulty of EFdA- terminated viral DNA to translocate. Thus, EFdA is termed a nucleotide reverse transcriptase translocation inhibitor (NRTTI). However, the inhibition mechanism of EFdA in HIV-infected cells (primary or cell lines) remains unknown. Toward that end, Co-I Malim has established an innovative assay that enables study of the EFdA antiviral mechanism in HIV-infected cells. Additionally, in vitro passage experiments have identified mutations that impart EFdA resistance through two paradigm-shifting dual mechanisms or resistance: decreased incorporation/enhanced excision and decreased incorporation/enhanced translocation. Data on an EFdA analog suggest efficient inhibition of EFdA-resistant HIV, although the mechanism of this phenomenon is not understood. Notably, treatment with key new generation NNRTI doravirine (DOR) leads to resistance mutations that impart hypersusceptibility to EFdA, paving the way for EFdA/DOR combination therapies. We hypothesize that the structural attributes of EFdA and its analogs impact interactions with diverse RTs (wild-type, drug- resistant, from diverse clades, viruses), leading to clinically important differences in efficiency of inhibition, resistance, hypersusceptibility, and biochemical mechanism of action. This hypothesis will be tested through three specific aims, which endeavor to answer the above questions using a combination of novel sequencing, biochemical, structural, and virological approaches. This work will help optimize combination therapies that reduce drug burden and have exceptional long-acting potential, addressing adherence challenges of chronic HIV treatment.

Some references relevant to our EFdA-related work:

https://projectreporter.nih.gov/project_info_results.cfm?aid=9459305&icde=45906984

Targeting HIV RNase H, the ‘final frontier’

CA is a structural protein essential for early and late events of HIV replication. Its multiple roles in infection and pathogenesis are not entirely understood. CA is an attractive therapeutic target since proper capsid formation is required for virus infectivity. We have discovered 18E8 (unpublished, to be patented), a compound with antiviral properties that interferes with the rate of CA multimerization. We have shown 18E8 to work by a novel mechanism and recently received an NIH grant to study this mechanism. Importantly, during this study we have solved the crystal structure of native hexameric full length HIV CA, an elusive structure for ~20 years. The structure provides insights into HIV biology and has resulted into collaborations with distinguished scientists in the field. Future efforts focus on providing the molecular details of CA interactions with cellular host proteins and improving our early inhibitors towards the development of innovative CA-targeting HIV therapeutics.

Discovery and characterization of a new mechanism of HIV multi-drug resistance

In collaboration with Japanese clinical scientists and independently from competing groups we helped demonstrate that RT mutation N348I causes resistance to drugs that belong to multiple classes of antiretrovirals (PI: Eiichi Kodama) (Hachiya et al. 2008). Our findings contributed to the redesign of clinical tools that detect HIV resistance mutations in patients and define the course of therapies. We also discovered new RT mutati on K70Q that causes multidrug resistance to tenofovir-based regimens (Hachiya et al, 2011). Ongoing and future efforts focus on the study of polymorphisms in various HIV clades towards elucidating drug resistance differences among various HIV subtypes, obtaining knowledge that should help optimize treatment strategies.

Novel methods for identification of drug resistance in HIV and HBV patients

In a related effort on drug resistance, we accepted an invitation for collaboration by Genematrix, a publicly traded Korean company, which manufactures mass spectrometry instrumentation. Based on our experience with drug resistance in HIV and other viruses Genematrix and ourgroup published a method that increases the sensitivity of current drug-resistance detection protocols, so that we can detect earlier drug resistance mutations and accordingly direct changes in therapeutic regimens (funding by 5-yr grant from the Korean government/Genematrix).

Discovery of FMDV inhibitors

FMDV is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock. We discovered and characterized a novel inhibitor of FMDV replication. Through collaboration with USDA scientists at Plum Island NY (Dr. E. Rieder and colleagues) we demonstrated antiviral activity of the compound (Durk et al, 2010), and now have an international patent for its use. We have shown that the novel targeted pocket is conserved in other viruses which we are currently studying.

Discovery of multiple assays for optimal combinations of HCV inhibitors

We have recently started a project on HCV in collaboration with world-leader in the field Dr. Charles Rice (Rockefeller University) and obtained 5-yr NIH funding R01AI099284. We have established novel microscopy-based assays for identifying optimal combinations of anti-HCV drugs. We are using these novel technologies to address the effect of host factors in the HCV life cycle. These studies were initiated by research faculty in our lab Dr. R. Ralston, who is now a Research Professor at Kansas Univ. Medical Center.