Professor of Biochemistry
Director of the Biological Chemistry Program
B.A. Cornell University
M.D./Ph.D. Stanford University
Michael Kay's Lab Page
Michael Kay's PubMed Literature Search
Molecular Biology Program
Biological Chemistry Program
Protein Design, Viral Entry, and Synthetic Biology
Our lab focuses on mirror-image peptides and proteins, which have great therapeutic potential because of their resistance to proteolysis. Our primary biological interest is developing D-peptide inhibitors of viral entry, particularly for the prevention and treatment of HIV and Ebola, though we are now expanding into diverse therapeutic areas including inflammation and cancer.
Mirror-image (D-peptide) Inhibitors
D-peptides are composed of mirror-image D-amino acids and have many potential advantages as therapeutics including low immunogenicity and protease resistance (allowing them to last much longer in the body than natural peptides). Since there are no natural examples to guide D-peptide design, we employ a high-throughput screening method called mirror-image phage display. In traditional phage display, large peptide libraries are ‘displayed’ on the bacteriophage surface (each phage bearing a different peptide). High-affinity binders are identified through several rounds of screening against a specific target. In mirror-image phage display, the target of interest is synthesized from D-amino acids to produce a mirror-image target. L-peptides displayed on phage are then selected for binding to the D-target. By symmetry, D-versions of the discovered peptides will bind to the natural L-target (see figure).
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 CD4/coreceptor engagement, which triggers gp41 to extend and 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 D-peptide inhibitors that bind to this intermediate and prevent HIV fusion and entry. The durability of D-peptides is particularly suitable for use as a microbicide, a topical preventative agent. Our inhibitor design also incorporates a unique "resistance capacitor" that provides a reserve of binding energy to combat potential resistance mutations (a major problem with HIV drugs).
Our most advanced D-peptide inhibitor of HIV entry, PIE12-trimer, potently inhibits all major HIV strains and has a high barrier to the emergence of viral resistance. A focus of the lab is understanding how HIV ultimately develops resistance to this inhibitor. PIE12-trimer is a promising candidate both for prevention and treatment of HIV (preclinical studies are underway in collaboration with local biotech company Navigen). See figure for a model of PIE12-trimer binding to the trimeric coiled coil.
Inhibiting Ebola Entry
Ebola is an enveloped, negative-strand RNA virus that causes severe hemorrhagic fever with a high mortality rate. 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 inhibition of the pre-hairpin intermediate. Guided by our HIV experience, we are creating peptide mimics of the Ebola fusion protein trimeric coiled coil region and using these mimics as targets in mirror-image phage display.
The D. coli Project
Currently, D-peptide discovery efforts are limited to small target proteins that can be synthesized using solid-phase peptide synthesis (SPPS) (typically <100 residues). Our lab is developing tools to create larger chemically synthesized proteins and investigating mechanisms for folding these increasingly complex proteins. Recently, we chemically synthesized a record-length (312-residue) protein, DapA. Under physiologic conditions, DapA cannot fold on its own and requires the assistance of a bacterial chaperone (GroEL/ES). Using a synthetic version of D-DapA, we showed that GroEL/ES is ‘ambidextrous’, capable of folding mirror-image DapA. This result suggests that natural chaperones can be used to fold D-peptides for mirror-image drug discovery and synthetic biology applications.
Looking ahead, we are working to make D-protein synthesis routine by developing new tools that facilitate chemical protein synthesis. Such tools will enable us to pursue larger target proteins for mirror-image drug development. Our long-term goals are to synthesize a D-ribosome and ultimately create a fully synthetic mirror-image organism that we have dubbed “D. coli”.
- Jacobsen MJ, Petersen ME, Ye X, Galibert M, Lorimer GH, Aucagne V, and Kay MS. A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis (2016). J Am Chem Soc. 138(36):11775-82.
- Petersen ME, Jacobsen MT, Kay MS. Synthesis of tumor necrosis factor α for use as a mirror-image phage display target (2016). Org Biomol Chem. 14(23):5298-303.
- Clinton TR, Weinstock MT, Jacobsen MT, Szabo-Fresnais N, Pandya MJ, Whitby FG, Herbert AS, Prugar LI, McKinnon R, Hill CP, Welch BD, Dye JM, Eckert DM, Kay MS. Design and characterization of ebolavirus GP prehairpin intermediate mimics as drug targets (2015). Protein Sci. 24:446-63.
- Weinstock MT, Jacobsen MT, and Kay MS. Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity (2014). PNAS 111: 11679–11684.
- Mesquita PM, Srinivasan P, Johnson TJ, Rastogi R, Evans-Strickfaden T, Kay MS, Buckheit KW, Buckheit RW Jr, Smith JM, Kiser PF, Herold BC. Novel preclinical models of topical PrEP pharmacodynamics provide rationale for combination of drugs with complementary properties (2013). Retrovirology 10:113.
- Pang HB, Hevroni L, Kol N, Eckert DM, Tsvitov M, Kay MS*, Rousso I*. Virion Stiffness Regulates Immature HIV-1 Entry (2013). Retrovirology 10:4.
- Francis, JN, Redman, JS, Eckert, DM, and Kay MS. Design of a Modular Tetrameric Scaffold for the Synthesis of Membrane-Localized D-peptide Inhibitors of HIV-1 Entry (2012). Bioconjugate Chemistry 23: 1252-8.
- Weinstock, MT, Francis, JN, Redman, JS, and Kay, MS (2012). Protease-Resistant Peptide Design – Empowering Nature’s Fragile Warriors Against HIV. Peptide Science, 98: 431-42.