The D. coli Project
The presence of stereogenic centers endows biomolecules with chirality, making the existence of mirror-image stereoisomers (enantiomers) chemically possible. All known living organisms use nucleic acids containing D-ribose/deoxyribose (RNA/DNA) and proteins composed of L-amino acids. Our lab develops D-peptide inhibitors of viral entry. As the mirror-image of naturally occurring peptides, D-peptides are not degraded by natural proteases, leading to a longer in vivo half-life and reduced immunogenicity. Currently, D-peptide discovery efforts are limited based on the size of target that can be readily synthesized by solid-phase peptide synthesis (SPPS).
Our lab is breaking the barriers of SPPS to create larger and larger chemically synthesized peptides and investigating mechanisms for folding these increasingly complex proteins. Recently, we chemically synthesized a record-length protein, the 312-residue bacterial protein DapA, in both L and D forms. Under physiologic conditions, DapA requires the GroEL/ES chaperone to fold. With our two versions of DapA, we showed GroEL/ES is ‘ambidextrous’, capable of folding both L- and D-DapA. Therefore, natural chaperones are likely to be valuable tools for the folding of D-peptides for mirror-image drug discovery and synthetic biology applications.
We are also developing computational tools to more efficiently design SPPS projects. Our Automated Ligator (Aligator) program predicts the optimal peptide segments needed to prepare entire proteins. As Aligator removes the tedious, costly manual chemical protein synthesis design process, we envision Aligator to be an important tool in creating novel large and/or multi-protein complexes.
Ultimately, we hope to overcome the SPPS limits by assembling a mirror-image in vitro translation apparatus (D-ribosome) and eventually a mirror-image organism, an effort that we have dubbed the “D. coli” project.
D-peptide Inhibitors of HIV Entry
HIV membrane fusion (mediated by the HIV gp41/gp120 complex) has been identified as a promising target for inhibition. Fusion is initiated by contact with a CD4+ target cell, which triggers a conformational change in gp41. gp41 extends to lance the target cell before collapsing into a six-helix “trimer of hairpins” that pulls the viral and target membranes together, leading to fusion. During this conformational transition, gp41 forms a transient pre-hairpin intermediate composed of a trimeric coiled coil. We are developing peptide and protein inhibitors that bind to this intermediate and prevent the progression of HIV fusion and entry.
In particular, we are interested in discovering small D-amino acid peptides that inhibit HIV membrane fusion and entry using mirror-image phage display. In this technique the target of interest is synthesized from D-amino acids. L-peptides displayed on phage are selected for binding to the D-target. By symmetry, D-versions of the discovered peptides will bind to the natural L-target. D-peptides have many potential advantages as therapeutics including low immunogenicity and protease resistance. These peptides also allow us to study for the first time the nature of high affinity interactions between L and D-peptides.
Our most advanced D-peptide inhibitor of HIV entry, PIE12-trimer, potently inhibits all clinically relevant strains of HIV and possesses a very high barrier to the emergence of viral resistance. PIE12-trimer is a promising candidate both for prevention and treatment of HIV. In collaboration with a local biotech company, Navigen, we are performing preclinical studies of PIE12-trimer.
Inhibiting Ebola Entry
Ebola is an enveloped, negative-strand RNA virus that causes severe hemorrhagic fever with an overall mortality rate approaching 70%. In 2014-2015, an alarming large western African outbreak has covered a wide geographic area and crossed international borders. Because of ease of transmission, high mortality, and lack of treatment or prevention options, the CDC places Ebola in its highest category of potential agents of bioterrorism. There is a vital need for a preventative and/or therapeutic to protect against future natural, accidental or deliberate outbreaks.
Although Ebola and HIV belong to distinct viral families and possess different morphology and genome structure, they use a common mechanism of entry into their host cells and are likely vulnerable to similar inhibition strategies of targeting the pre-hairpin intermediate. Using our HIV program as a model, we are creating peptide mimics of the Ebola fusion protein (GP2) trimeric coiled coil region and using these mimics as targets in mirror-image phage display.