Prevention and treatment of SHIVAD8 infection in rhesus macaques by a potent d-peptide HIV entry inhibitor.

Cholesterol-PIE12-trimer (CPT31) is a potent d-peptide HIV entry inhibitor that targets the highly conserved gp41 N-peptide pocket region. CPT31 exhibited strong inhibitory breadth against diverse panels of primary virus isolates. In a simian-HIV chimeric virus AD8 (SHIVAD8) macaque model, CPT31 prevented infection from a single high-dose rectal challenge. In chronically infected animals, CPT31 monotherapy rapidly reduced viral load by ∼2 logs before rebound occurred due to the emergence of drug resistance. In chronically infected animals with viremia initially controlled by combination antiretroviral therapy (cART), CPT31 monotherapy prevented viral rebound after discontinuation of cART. These data establish CPT31 as a promising candidate for HIV prevention and treatment.

Pharmacokinetic and Chemical Synthesis Optimization of a Potent d-Peptide HIV Entry Inhibitor Suitable for Extended-Release Delivery.

Peptides often suffer from short in vivo half-lives due to proteolysis and renal clearance that limit their therapeutic potential in many indications, necessitating pharmacokinetic (PK) enhancement. d-Peptides, composed of mirror-image d-amino acids, overcome proteolytic degradation but are still vulnerable to renal filtration due to their small size. If renal filtration could be slowed, d-peptides would be promising therapeutic agents for infrequent dosing, such as in extended-release depots. Here, we tether a diverse set of PK-enhancing cargoes to our potent, protease-resistant d-peptide HIV entry inhibitor, PIE12-trimer. This inhibitor panel provides an opportunity to evaluate the PK impact of the cargoes independently of proteolysis. While all the PK-enhancing strategies (PEGylation, acylation, alkylation, and cholesterol conjugation) improved in vivo half-life, cholesterol conjugation of PIE12-trimer dramatically improves both antiviral potency and half-life in rats, making it our lead anti-HIV drug candidate. We designed its chemical synthesis for large-scale production (CPT31) and demonstrated that the PK profile in cynomolgous monkeys supports future development of monthly or less frequent depot dosing in humans. CPT31 could address an urgent need in both HIV prevention and treatment.

On the stability of parainfluenza virus 5 F proteins.

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

Design and characterization of ebolavirus GP prehairpin intermediate mimics as drug targets.

Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify D-peptide inhibitors of ebolavirus entry.

Probing the functions of the paramyxovirus glycoproteins F and HN with a panel of synthetic antibodies.

Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction. Importance: The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells.

Structure of the parainfluenza virus 5 (PIV5) hemagglutinin-neuraminidase (HN) ectodomain.

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a "4-heads-down" conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a "2-heads-up/2-heads-down" conformation where two heads (covalent dimers) are in the "down position," forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an "up position." The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.

Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering.

The Paramyxoviridae family of enveloped viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating virus-cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxovirus parainfluenza virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoviruses, varying energy requirements for fusion activation, F activation involving the parainfluenza virus 5 HN stalk domain, and properties of a chimeric paramyxovirus HN protein, we propose a simple model for the activation of paramyxovirus fusion.

Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion.

Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 Å, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

One percent tenofovir applied topically to humanized BLT mice and used according to the CAPRISA 004 experimental design demonstrates partial protection from vaginal HIV infection, validating the BLT model for evaluation of new microbicide candidates.

Recent iPrEx clinical trial results provided evidence that systemic preexposure prophylaxis (PrEP) with emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) can partially prevent rectal HIV transmission in humans. Similarly, we have previously demonstrated that systemic administration of the same FTC-TDF combination efficiently prevented rectal transmission in humanized bone marrow/liver/thymus (BLT) mice. The CAPRISA 004 trial recently demonstrated that topical application of the tenofovir could partially prevent vaginal HIV-1 transmission in humans. To further validate the usefulness of the BLT mouse model for testing HIV prevention strategies, we evaluated the topical administration of tenofovir as used in CAPRISA 004 to prevent vaginal HIV transmission in BLT mice. Our results demonstrate that vaginally administered 1% tenofovir significantly reduced HIV transmission in BLT mice (P = 0.002). Together with the results obtained after systemic antiretroviral PrEP, these topical inhibitor data serve to validate the use of humanized BLT mice to evaluate both systemic and topical inhibitors of HIV transmission. Based on these observations, we tested six additional microbicide candidates for their ability to prevent vaginal HIV transmission: a C-peptide fusion inhibitor (C52L), a membrane-disrupting amphipathic peptide inhibitor (C5A), a trimeric d-peptide fusion inhibitor (PIE12-Trimer), a combination of reverse transcriptase inhibitors (FTC-TDF), a thioester zinc finger inhibitor (TC247), and a small-molecule Rac inhibitor (NSC23766). No protection was seen with the Rac inhibitor NSC23766. The thioester compound TC247 offered partial protection. Significant protection was afforded by FTC-TDF, and complete protection was offered by three different peptide inhibitors tested. Our results demonstrate that these effective topical inhibitors have excellent potential to prevent vaginal HIV transmission in humans.

Design of a potent D-peptide HIV-1 entry inhibitor with a strong barrier to resistance.

The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific D-peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. D-peptides (peptides composed of D-amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first D-peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.

Characterization of the steric defense of the HIV-1 gp41 N-trimer region.

During viral entry, HIV gp41 adopts a transient conformation called the "prehairpin intermediate" in which a highly conserved therapeutic target, the N-trimer, is exposed. Despite extensive discovery efforts, potent and broadly neutralizing antibodies that target the N-trimer are elusive. We previously demonstrated the N-trimer is protected by a steric block that prevents large proteins, such as antibodies, from accessing it. Here we further characterize the steric block and identify its source. To study the N-trimer steric accessibility, we produced two sets of C-peptide inhibitors (a potent inhibitor targeting the N-trimer) fused to cargo proteins of increasing size facing either the virus or cell side of the prehairpin intermediate. Both bulky inhibitor sets show a steric block, but the effect is more pronounced with virus-side cargo. Additionally, both sets maintain their potencies in a modified entry assay that removes possible sources of target cell steric hindrance. These results implicate a viral source, likely gp120, as the primary component of the steric block. In addition, we studied the steric accessibility of the "pocket" region of the N-trimer, a highly attractive drug and vaccine target. We demonstrated a pocket-specific antibody, D5, is more potent as an scFv than as a full-length IgG, suggesting the N-trimer steric restriction extends to the pocket. This characterization will facilitate the design of sterically restricted antigens that mimic the steric environment of the N-trimer in the prehairpin intermediate and are capable of inducing potent and broadly neutralizing antibodies that circumvent the N-trimer steric block.

Potent D-peptide inhibitors of HIV-1 entry.

During HIV-1 entry, the highly conserved gp41 N-trimer pocket region becomes transiently exposed and vulnerable to inhibition. Using mirror-image phage display and structure-assisted design, we have discovered protease-resistant D-amino acid peptides (D-peptides) that bind the N-trimer pocket with high affinity and potently inhibit viral entry. We also report high-resolution crystal structures of two of these D-peptides in complex with a pocket mimic that suggest sources of their high potency. A trimeric version of one of these peptides is the most potent pocket-specific entry inhibitor yet reported by three orders of magnitude (IC(50) = 250 pM). These results are the first demonstration that D-peptides can form specific and high-affinity interactions with natural protein targets and strengthen their promise as therapeutic agents. The D-peptides described here address limitations associated with current L-peptide entry inhibitors and are promising leads for the prevention and treatment of HIV/AIDS.

Inhibiting HIV fusion with a beta-peptide foldamer.

Linear peptides derived from the HIV gp41 C-terminus (C-peptides), such as the 36-residue Fuzeon, are potent HIV fusion inhibitors. These molecules bind to the N-peptide region of gp41 and inhibit an intramolecular protein-protein interaction that powers fusion of the viral and host cell membranes. The N-peptide region contains a surface pocket that is occupied in the post-fusion state by three alpha-helical residues found near the gp41 C-terminus: Trp628, Trp631, and Ile635-the WWI epitope. Here, we describe a set of beta3-decapeptides (betaWWI-1-4) in which the WWI epitope is presented on one face of a short 14-helix stabilized by macrodipole neutralization and side chain-side chain salt bridges. betaWWI-1-4 bind in vitro to IZN17, a validated gp41 model, and inhibit syncytia formation in cell culture. Molecules lacking a complete WWI functional epitope neither bind IZN17 nor inhibit syncytia formation. These results provide evidence that short beta-peptide 14-helices can inhibit an intramolecular protein-protein interaction in vivo. Molecules related to betaWWI-1-4 could represent starting points for the development of highly potent inhibitors or antigens effective against HIV or other viruses, including SARS, Ebola, HRSV, and influenza, that employ common fusion mechanisms.

Steric accessibility of the HIV-1 gp41 N-trimer region.

During human immunodeficiency virus entry, gp41 undergoes a series of conformational changes that induce membrane fusion. Immediately prior to fusion, gp41 exists in a prehairpin intermediate in which the N- and C-peptide regions of gp41 are exposed. Rearrangement of this intermediate into a six-helix bundle composed of a trimeric coiled coil from the N-peptide region (N-trimer) surrounded by three peptides from the C-peptide region provides the driving force for membrane fusion, whereas prevention of six-helix bundle formation inhibits viral entry. Because of its central role in mediating viral entry, the N-trimer region of gp41 is a key vaccine target. Extensive efforts to discover potent and broadly neutralizing antibodies (Abs) against the N-trimer region have, thus far, been unsuccessful. In this study, we attached a potent C-peptide inhibitor that binds to the N-trimer region to cargo proteins of various sizes to examine the steric accessibility of the N-trimer during fusion. These inhibitors show a progressive loss of potency with increasing cargo size. Extension of the cargo/C-peptide linker partially restores inhibitory potency. These results demonstrate that the human immunodeficiency virus defends its critical hairpin-forming machinery by steric exclusion of large proteins and may explain the current dearth of neutralizing Abs against the N-trimer. In contrast, previous results suggest the C-peptide region is freely accessible during fusion, demonstrating that the N- and C-peptide regions are in structurally distinct environments. Based on these results, we also propose new strategies for the generation of neutralizing Abs that overcome this steric block.

Ubiquitin interactions of NZF zinc fingers.

Ubiquitin (Ub) functions in many different biological pathways, where it typically interacts with proteins that contain modular Ub recognition domains. One such recognition domain is the Npl4 zinc finger (NZF), a compact zinc-binding module found in many proteins that function in Ub-dependent processes. We now report the solution structure of the NZF domain from Npl4 in complex with Ub. The structure reveals that three key NZF residues (13TF14/M25) surrounding the zinc coordination site bind the hydrophobic 'Ile44' surface of Ub. Mutations in the 13TF14/M25 motif inhibit Ub binding, and naturally occurring NZF domains that lack the motif do not bind Ub. However, substitution of the 13TF14/M25 motif into the nonbinding NZF domain from RanBP2 creates Ub-binding activity, demonstrating the versatility of the NZF scaffold. Finally, NZF mutations that inhibit Ub binding by the NZF domain of Vps36/ESCRT-II also inhibit sorting of ubiquitylated proteins into the yeast vacuole. Thus, the NZF is a versatile protein recognition domain that is used to bind ubiquitylated proteins during vacuolar protein sorting, and probably many other biological processes.

P2Y2 receptor-stimulated release of prostaglandin E2 by rat inner medullary collecting duct preparations.

Extracellular nucleotides, acting through the P2Y2 receptor and the associated phosphoinositide-Ca2+ signaling pathway, inhibit AVP-stimulated osmotic water permeability in rat inner medullary collecting duct (IMCD). Because a rise in intracellular Ca2+ is frequently associated with enhanced arachidonic acid metabolism, we examined the effect of activation of the P2Y2 receptor on release of PGE2 in freshly prepared rat IMCD suspensions. Unstimulated IMCD released moderate, but significant, amounts of PGE2, which were more sensitive to cyclooxygenase (COX)-2 than COX-1 inhibition. Agonist activation of P2Y2 receptor by adenosine 5'-O-(3-thiotriphosphate) enhanced release of PGE2 from IMCD in a time- and concentration-dependent fashion. Purinergic-stimulated release of PGE2 was completely blocked by nonspecific COX inhibitors (flurbiprofen and 2-acetoxyphenylhept-2-ynyl sulfide). Differential COX inhibition studies revealed that purinergic-stimulated release of PGE2 was more sensitive to a COX-1-specific inhibitor (valeroyl salicylate) than a COX-2-specific inhibitor (NS-398). Thus purinergic stimulation resulted in significantly more release of PGE2 in the presence of COX-2 inhibitor than COX-1 inhibitor. If it is assumed that increased release of PGE2 is related to its increased production, our results suggest that purinergic stimulation of IMCD results in enhanced production and release of PGE2 in a COX-1-dependent fashion. Because PGE2 is known to affect transport of water, salt, and urea in IMCD, interaction of the purinergic system with the prostanoid system in IMCD can modulate handling of water, salt, and urea by IMCD and, thus, may constitute an AVP-independent regulatory mechanism.