Project Summary
The 21st century has seen the emergence of multiple lethal human coronaviruses (SARS-CoV, MERS-CoV,
and now SARS-CoV-2). There is an urgent need for therapeutic options to combat the current and inevitable
future SARS-like pandemics. Coronaviruses infect cells using a conserved entry mechanism shared by viruses
across multiple families (including HIV, Ebola, and influenza) in which two regions of the trimeric viral spike
protein (HR1 and HR2) collapse to form a highly stable six-helix bundle structure that forces the viral and
cellular membranes together, inducing membrane fusion. Inhibitor binding to HR1 blocks six-helix bundle
formation and stops viral entry, preventing infection. Our lab specializes in mirror-image phage display (MIPD),
an innovative approach to identify novel synthetic protease-resistant D-peptide drug candidates, with a special
focus on the inhibition of viral entry (with our HIV-1 drug, CPT31, set to begin clinical trials). D-peptides
(peptides composed of mirror-image D-amino acids) cannot be digested by proteases in the body and,
therefore, possess significant therapeutic advantages including extended half-life, lower dosing, reduced
immunogenicity (not digested for MHC presentation), and durability in protease-rich environments such as the
respiratory tract. To address the current health crisis, we are expediting our drug discovery process to identify
D-peptide entry inhibitors that target the conserved HR1 of SARS-related coronaviruses. We have designed,
synthesized, and characterized our HR1 mimic drug targets and are using them in MIPD to identify D-peptide
inhibitors of 6-helix bundle formation and viral entry.
In this proposal, we will chemically synthesize the D-peptides identified by MIPD and characterize their
target affinity (using surface plasmon resonance) and antiviral activity against SARS-CoV and SARS-CoV-2
pseudoviruses. Promising D-peptides will be affinity-matured using a second round of MIPD to optimize
potency. Using our custom-designed PEG scaffold (the backbone of CPT31), we will trimerize the highest
affinity D-peptide candidates to improve avidity for the trimeric spike target and attach a membrane-localizing
group, such as cholesterol, that will enrich the D-peptide at the cellular site of viral entry and improve in vivo
half-life. These leading D-peptides will be tested against authentic virus (in collaboration with USU's Institute
for Antiviral Research). Our objective is to have one D-peptide candidate with =100 nM in vitro EC90 against
SARS-CoV-2 and SARS and a good therapeutic index (EC50/CC50 >100) to advance to in vivo PK and efficacy
studies, using USU's hamster model of SARS-CoV-2 infection. At the end of the grant period, we expect to
have one D-peptide lead with demonstrated in vivo animal efficacy, poised for IND-enabling preclinical studies
and development as a SARS-related coronavirus treatment and/or preventative.