Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities.

Large peptide biosynthesis is a valuable alternative to conventional chemical synthesis. Enfuvirtide, the largest therapeutic peptide used in HIV infection treatment, was synthesized in our thermostable chaperone-based peptide biosynthesis system and evaluated for peptide quality as well as the profile of process-related impurities. Host cell proteins (HCPs) and BrCN cleavage-modified peptides were evaluated by LC-MS in intermediate. Cleavage modifications during the reaction were assessed after LC-MS maps were aligned by simple in-house algorithm and formylation/oxidation levels were estimated. Circular dichroism spectra of the obtained enfuvirtide were compared to the those of the chemically- synthesized standard product. Final-product endotoxin and HCPs content were assessed resulting 1.06 EU/mg and 5.58 ppm respectively. Peptide therapeutic activity was measured using the MT-4 cells HIV infection-inhibition model. The biosynthetic peptide IC50 was 0.0453 µM while the standard one had 0.0180 µM. Non-acylated C-terminus was proposed as a cause of IC50 and CD spectra difference. Otherwise, the peptide has met all the requirements of the original chemically synthesized enfuvirtide in the cell-culture and in vivo experiments.

The Glu143 Residue Might Play a Significant Role in T20 Peptide Binding to HIV-1 Receptor gp41: An In Silico Study.

Despite the enormous efforts made to develop other fusion inhibitors for HIV, the enfuvirtide (known as T20) peptide is the only approved HIV-1 inhibitory drug so far. Investigating the role of potential residues of the T20 peptide's conformational dynamics could help us to understand the role of potential residues of the T20 peptide. We investigated T20 peptide conformation and binding interactions with the HIV-1 receptor (i.e., gp41) using MD simulations and docking techniques, respectively. Although the mutation of E143 into alanine decreased the flexibility of the E143A mutant, the conformational compactness of the mutant was increased. This suggests a potential role of E143 in the T20 peptide's conformation. Interestingly, the free energy landscape showed a significant change in the wild-type T20 minimum, as the E143A mutant produced two observed minima. Finally, the docking results of T20 to the gp41 receptor showed a different binding interaction in comparison to the E143A mutant. This suggests that E143 residue can influence the binding interaction with the gp41 receptor. Overall, the E143 residue showed a significant role in conformation and binding to the HIV-1 receptor. These findings can be helpful in optimizing and developing HIV-1 inhibitor peptides.

Virus Entry Inhibitors: Past, Present, and Future.

The approval of enfuvirtide marked a milestone for the development of virus entry inhibitor-based antiviral therapeutics. Since then, more peptide-, small-molecule-, and protein-based entry inhibitors have been identified and approved for viral diseases. Here we reviewed the development of virus entry inhibitors and the advantages and disadvantages of peptide-, small-molecule-, and protein-based entry inhibitors, herein summarizing the future trend of these antivirals. Virus entry inhibitors take effect outside the host cell, making them good candidates for development as pre- and post-exposure prophylaxis, microbicides, and therapeutics. This chapter, as well as this book, provides more information on the development and modification of peptide-, small-molecule-, and protein-based virus entry inhibitors.

Massively Parallel Profiling of HIV-1 Resistance to the Fusion Inhibitor Enfuvirtide.

Identifying drug resistance mutations is important for the clinical use of antivirals and can help define both a drug's mechanism of action and the mechanistic basis of resistance. Resistance mutations are often identified one-at-a-time by studying viral evolution within treated patients or during viral growth in the presence of a drug in cell culture. Such approaches have previously mapped resistance to enfuvirtide, the only clinically approved HIV-1 fusion inhibitor, to enfuvirtide's binding site in the N-terminal heptad repeat (NHR) of the Envelope (Env) transmembrane domain as well as a limited number of allosteric sites. Here, we sought to better delineate the genotypic determinants of resistance throughout Env. We used deep mutational scanning to quantify the effect of all single-amino-acid mutations to the subtype A BG505 Env on resistance to enfuvirtide. We identified both previously characterized and numerous novel resistance mutations in the NHR. Additional resistance mutations clustered in other regions of Env conformational intermediates, suggesting they may act during different fusion steps by altering fusion kinetics and/or exposure of the enfuvirtide binding site. This complete map of resistance sheds light on the diverse mechanisms of enfuvirtide resistance and highlights the utility of using deep mutational scanning to comprehensively map potential drug resistance mutations.

A 30 kDa polyethylene glycol-enfuvirtide complex enhances the exposure of enfuvirtide in lymphatic viral reservoirs in rats.

HIV therapy with anti-retroviral drugs is limited by the poor exposure of viral reservoirs, such as lymphoid tissue, to these small molecule drugs. We therefore investigated the effect of PEGylation on the anti-retroviral activity and subcutaneous lymphatic pharmacokinetics of the peptide-based fusion inhibitor enfuvirtide in thoracic lymph duct cannulated rats. Both the peptide and the PEG were quantified in plasma and lymph via ELISA. Conjugation to a single 5 kDa linear PEG decreased anti-HIV activity three-fold compared to enfuvirtide. Whilst plasma and lymphatic exposure to peptide mass was moderately increased, the loss of anti-viral activity led to an overall decrease in exposure to enfuvirtide activity. A 20 kDa 4-arm branched PEG conjugated with an average of two enfuvirtide peptides decreased peptide activity by six-fold. Plasma and lymph exposure to enfuvirtide, however, increased significantly such that anti-viral activity was increased two- and six-fold respectively. The results suggest that a multi-enfuvirtide-PEG complex may optimally enhance the anti-retroviral activity of the peptide in plasma and lymph.

Evolved Proteins Inhibit Entry of Enfuvirtide-Resistant HIV-1.

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.

HIV-1 inhibitory properties of eCD4-Igmim2 determined using an Env-mediated membrane fusion assay.

Human Immunodeficiency Virus-1 (HIV-1) entry is dependent on the envelope glycoprotein (Env) that is present on the virion and facilitates fusion between the envelope and the cellular membrane. The protein consists of two subunits, gp120 and gp41, with the former required for binding the CD4 receptor and either the CXCR4 or CCR5 coreceptor, and the latter for mediating fusion. The requirement of fusion for infection has made Env an attractive target for HIV therapy development and led to the FDA approval of enfuvirtide, a fusion inhibitor. Continued development of entry inhibitors is warranted because enfuvirtide resistant HIV-1 strains have emerged. In this study, a novel HIV-1 fusion assay was validated using neutralizing antibodies and then used to investigate the mechanism of action of eCD4-Igmim2, an HIV-1 inhibitor proposed to cooperatively bind the CD4 binding site and the sulfotyrosine-binding pocket of gp120. Greater reduction in fusion levels was observed with eCD4-Igmim2 in the fusion assay than all of the gp120 antibodies evaluated. Lab adapted isolates, HIV-1HXB2 and HIV-1YU2, were sensitive to eCD4-Igmim2 in the fusion assay, while primary isolates, HIV-1BG505 and HIV-1ZM651 were resistant. These results correlated with greater IC50 values for primary isolates compared to the lab adapted isolates observed in a virus neutralization assay. Analysis of gp120 models identified differences in the V1 and V2 domains that are associated with eCD4-Igmim2 sensitivity. This study highlights the use of a fusion assay to identify key areas for improving the potency of eCD4-Igmim2.