Helix‐Grafted Pleckstrin Homology Domains Suppress HIV‐1 Infection of CD4‐Positive Cells

Tennyson, Rachel L.; Walker, Susanne; Ikeda, Terumasa; Harris, Reuben S.; Kennan, Alan J.; McNaughton, Brian R.

doi:10.1002/cbic.201600329

Cited by 3 publications

(5 citation statements)

References 27 publications

(30 reference statements)

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“…[16] We next sought to evaluate sequence variability in the grafted helix. Five positions on Cpep-ELMO (Figure 2a) that bury into the C-peptide helix-binding cleft of N-peptide trimer (wild-type sequence WWIYI) were randomized by saturation mutagenesis.…”

Section: Resultsmentioning

confidence: 99%

“…Having demonstrated that some of our evolved C-peptide ELMO helix-grafted display proteins express better than Cpep-ELMO, retain helical structure, and bind to gp41 5-helix in vitro , we next measured their ability to suppress HIV infection using a live virus assay we have previously reported[16, 23]. In this method, HIV IIIB is administered to CD4+ mammalian cells stably integrated with a plasmid that encodes the HIV-1 long-terminal repeat (LTR) upstream of green fluorescent protein.…”

Section: Resultsmentioning

confidence: 99%

“…[16, 17] A stable scaffold protein displaying a helix, which contains at least one solvent-exposed face, serves as a generic canvas upon which to paint, or extend, any desired helical interface. We term this method ‘helix-grafted display’ (Figure.…”

Section: Introductionmentioning

confidence: 99%

“…[16, 17] This therapeutic activity is the result of inhibiting an intramolecular PPI between HIV-1 gp41 C-peptides (α-helix) and a trimer of HIV-1 gp41 N-peptides, which contain C-peptide α-helix-binding clefts. This PPI generates a ‘trimer-of-hairpin’ assembly that brings HIV and mammalian cell membranes into close proximity, leading to membrane fusion and HIV-1 entry (Figure 1b).…”

Section: Introductionmentioning

confidence: 99%

“…Cpep-ELMO binds tightly to gp41 5-helix ( K D ~ 90 nM) and suppresses entry of HIV-1 into Cluster of Differentiation 4 (CD4) positive human cells. [16] In contrast to its peptide counterpart (the FDA approved peptide drug Fuzeon ™ ), Cpep-ELMO is stable in human serum.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins

Tennyson

Walker

Ikeda

et al. 2018

Bioorganic & Medicinal Chemistry

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Many therapeutically-relevant protein-protein interactions (PPIs) have been reported that feature a helix and helix-binding cleft at the interface. Given this, different approaches to disrupting such PPIs have been developed. While short peptides (<15 amino acids) typically do not fold into a stable helix, researchers have reported chemical approaches to constraining helix structure. However, these approaches rely on laborious, and often expensive, chemical synthesis and purification. Our premise is that protein-based solutions that stabilize a therapeutically-relevant helix offer a number of advantages. In contrast to chemically constrained helical peptides, or minimal/miniature proteins, which must be synthesized (at great expense and labor), a protein can be expressed in a cellular system (like all current protein therapeutics). If selected properly, the protein scaffold can stabilize the therapeutically-relevant helix. We recently reported a protein engineering strategy, which we call “helix-grafted display”, and applied it to the challenge of suppressing HIV entry. We have reported helix-grafted display proteins that inhibit formation of an intramolecular PPI involving HIV gp41 C-peptide helix, and HIV gp41 N-peptide trimer, which contain C-peptide helix-binding clefts. Here, we used yeast display to screen a library of grafted C-peptide helices for N-peptide trimer recognition. Using ‘hits’ from yeast display library screening, we evaluated the effect helix mutations have on structure, expression, stability, function (target recognition), and suppression of HIV entry.2009 Elsevier Ltd. All rights reserved.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins

Tennyson

Walker

Ikeda

et al. 2018

Bioorganic & Medicinal Chemistry

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Antibody‐Recruitment as a Therapeutic Strategy: A Brief History and Recent Advances

et al. 2022

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Antibodies are a significant and growing sector within the global pharmaceutical industry. The popularity of antibodies as therapeutics derives from -at least in part -evolvable affinity for virtually any disease-relevant cell surface receptor, as well as unique immunotherapeutic mechanisms of action, including neutralization, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC). While advances in the large-scale expression and purification of therapeutic antibodies have been made, these remain costly and laborious tasks. Agents that redirect endogenous antibodies to target a pathogen or malignant cell obviate the need for new antibody discovery and production. Chimeric antibody-recruiting technologies consist of a target cell surface receptor binding domain, and an endogenous antibody-binding domain. By design, these agents bring endogenous antibodies to the surface of a target pathogen or diseased cell, which can result in targeted cytotoxicity by antibody-dependent mechanisms. This review highlights seminal contributions and recent advances in this growing and important therapeutic field. Current Limitations to the Broader Use of Antibodies as TherapeuticsTheir evolvability, incredible in vivo stability, and diverse mechanisms of action make antibodies well suited for use as [a] Dr.

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Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1

et al. 2019

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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 to >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.

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Helix‐Grafted Pleckstrin Homology Domains Suppress HIV‐1 Infection of CD4‐Positive Cells

Cited by 3 publications

References 27 publications

Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins

Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins

Antibody‐Recruitment as a Therapeutic Strategy: A Brief History and Recent Advances

Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1

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