Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1 | ACS

Here, we report that gp41 C-peptide helix-grafted Sac7d (Sac7d-Cpep) potently suppresses HIV-1 entry in a live virus assay (IC50 = 1.9–12.4 nM). Yea...
0 downloads 0 Views 1MB Size
Subscriber access provided by Washington University | Libraries

Article

Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1 Terumasa Ikeda, Rachel Tennyson, Susanne N. Walker, Reuben S. Harris, and Brian McNaughton ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00362 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1 Terumasa Ikeda2,3,4, Rachel L Tennyson1,4, Susanne N. Walker1,4, Reuben S. Harris2,3, and Brian R. McNaughton* 1Department

of Chemistry, Colorado State University, 200 W. Lake Street, Fort Collins, CO, USA

2Department

of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church Street, Minneapolis, MN, USA

3Howard 4These

Hughes Medical Institute, University of Minnesota, Minneapolis, MN, USA

authors contributed equally

*Correspondence: [email protected]

Keywords: protein engineering · biologics · protein evolution · HIV · entry inhibitor

Drugs that block HIV-1 entry are relatively limited. Enfuvirtide is a 36-residue synthetic peptide that targets gp41 and blocks viral fusion. However, Enfuvirtide-resistant HIV has been reported, and this peptide drug requires daily injection. Previously, we have reported helix-grafted display proteins, consisting of HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these biologics inhibit HIV-1 entry with relatively modest and varied potency (IC50 190 nM - >1 μM). Here, we report that gp41 C-peptide helix-grafted Sac7d (Sac7d-Cpep) potently suppresses HIV-1 entry in a live virus assay (IC50 1.9-12.4 nM). Yeast display sequence optimization of solvent exposed helix residues led to new biologics with improved expression in E. coli (a common biosimilar expression host), with no appreciable change in entry inhibition. Evolved proteins inhibit the entry of a clinically-relevant mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide-resistant. Fusion proteins designed for serum stability also potently suppress HIV-1 entry. Collectively, we report several evolved biologics that are functional against an Enfuvirtide-resistant strain and are designed for serum stability.

Individuals at high risk for HIV-1 infection often take pre-exposure prophylaxis (PrEP), a cocktail consisting of Emtricitabine and Tenofovir (both reverse transcriptase inhibitors).(1) For patients already infected with HIV-1, therapeutics are available that act on a number of proteins necessary for HIV-1 proliferation. For example, the principal components of combination antiretroviral therapy (cART) are typically inhibitors of viral protease and reverse transcriptase enzymes.(2) Both PrEP and cART require daily administration due to the relatively short in vivo lifetime of these smallmolecule drugs.(1-2) Given the stigma associated with HIV infection, there is a significant psychological barrier to ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 17

obtaining, storing, and taking—on a daily basis—HIV antiretroviral prophylaxis and therapeutics. Since PrEP and cART do not inhibit HIV-1 entry, even miniscule levels of replication-competent viral genome integration by intracellular HIV-1 can lead to the formation of latent viral reservoirs, which make a cure virtually impossible. Since inhibition of virus entry, by definition, stops viral genome integration, entry inhibition may be preferred to protect against HIV-1 infection and the formation of latent viral reservoirs. HIV infection is initiated by binding between HIV gp120 and the cluster of differentiation 4 (CD4) receptor on immune cells, such as T helper cells, monocytes, macrophages, and dendritic cells.(3) This recognition event is followed by a conformational change in HIV gp120, which exposes another HIV protein – viral glycoprotein gp41. The C-terminus of gp41 inserts into the HIV membrane, while the N-terminus inserts into the membrane of the engaged immune cell. Physical entry of the viral contents into the bound immune cell requires conformational collapse of prefusogenic N-peptide and C-peptide trimers into a “trimer-of-hairpins” assembly that promotes membrane fusion – a mandatory step for infection (Figure 1A).(4) Only one FDA approved HIV entry inhibitor (Enfuvirtide) targets gp41.(5) Enfuvirtide is a 36-residue synthetic peptide that binds pre-fusogenic gp41 N-peptide trimer, inhibiting formation of the “trimer-of-hairpins” assembly required for viral membrane fusion and HIV infection of a host immune cell (Figure 1A). Enfuvirtide is costly to synthesize, has poor solubility and bioavailability, is sensitive to serum and interstitial proteases (in vivo half-life is approximately 4 hours) and thus requires daily parenteral administration.(6) Long-lasting Enfuvirtide conjugates have been reported (7); however, they require additional chemical synthesis and purification. Since Enfuvirtide is a single peptide, mutation of HIV gp41 can lead to Enfuvirtide-resistance.(8) Researchers have reported D-peptides (9), mixed α/β peptides (10), and longer helical scaffolds that display C-peptide residues (11). However, all of these require laborious chemical synthesis and purification. We have reported a technique called ‘helix-grafted display’ (Figure 1B), a protein engineering-based solution to generate biologics that target therapeutically-relevant helix-binding clefts, such as the gp41 N-peptide trimer prefusogenic assembly.(12) We demonstrated Pleckstrin Homology domains (PH domains) as suitable scaffolds, since they are generally stable, express in E. coli (a common bioproduction host), and contain a solvent-exposed helix. Specific PH domain helix residues are mutated to mimic gp41 C-peptide helix. Once the native helix is utilized, the remaining gp41 Cpeptide helix is then genetically fused. The resulting protein mimics a folded gp41-C-peptide helix. We reported a firstgeneration C-terminal helix-grafted protein (GLUE-Cpep, (12b) that binds gp41 ‘5-helix’ (13), a surrogate for pre-fusogenic ACS Paragon Plus Environment

Page 3 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

N-peptide trimer, in vitro and in a cell-based co-purification assay. Our second-generation N-terminal helix-grafted protein (Cpep-ELMO, (12a) modestly inhibits HIV entry (IC50 >1 μM) in a live virus assay. More recently we showed that yeast display optimization of grafted residues on Cpep-ELMO led to proteins with improved inhibition of HIV entry in a live virus assay (IC50 190–770 nM).(14)

RESULTS AND DISCUSSION Helix-grafted Sac7d (Sac7d-Cpep) potently binds 5-helix in E. coli cell lysate. Previous studies suggested a relationship between the size of the helix-grafted display scaffold and HIV-1 entry inhibition: smaller scaffolds are more effective.(12a) We therefore set our sights on a miniature (