7 research outputs found
Macrocyclic Envelope Glycoprotein Antagonists that Irreversibly Inactivate HIV‑1 <i>before</i> Host Cell Encounter
We
derived macrocyclic HIV-1 antagonists as a new class of peptidomimetic
drug leads. Cyclic peptide triazoles (cPTs) retained the gp120 inhibitory
and virus-inactivating signature of parent PTs, arguing that cyclization
locked an active conformation. The six-residue cPT <b>9</b> (AAR029b)
exhibited submicromolar antiviral potencies in inhibiting cell infection
and triggering gp120 shedding that causes irreversible virion inactivation.
Importantly, cPTs were stable to trypsin and chymotrypsin compared
to substantial susceptibility of corresponding linear PTs
Injectable Granular Hydrogels Enable Avidity-Controlled Biotherapeutic Delivery
Protein therapeutics
represent a rapidly growing class
of pharmaceutical
agents that hold great promise for the treatment of various diseases
such as cancer and autoimmune dysfunction. Conventional systemic delivery
approaches, however, result in off-target drug exposure and a short
therapeutic half-life, highlighting the need for more localized and
controlled delivery. We have developed an affinity-based protein delivery
system that uses guest–host complexation between β-cyclodextrin
(CD, host) and adamantane (Ad, guest) to enable sustained localized
biomolecule presentation. Hydrogels were formed by the copolymerization
of methacrylated CD and methacrylated dextran. Extrusion fragmentation
of bulk hydrogels yielded shear-thinning and self-healing granular
hydrogels (particle diameter = 32.4 ± 16.4 μm) suitable
for minimally invasive delivery and with a high host capacity for
the retention of guest-modified proteins. Bovine serum albumin (BSA)
was controllably conjugated to Ad via EDC chemistry without affecting
the affinity of the Ad moiety for CD (KD = 12.0 ± 1.81 μM; isothermal titration calorimetry).
The avidity of Ad–BSA conjugates was directly tunable through
the number of guest groups attached, resulting in a fourfold increase
in the complex half-life (t1/2 = 5.07
± 1.23 h, surface plasmon resonance) that enabled a fivefold
reduction in protein release at 28 days. Furthermore, we demonstrated
that the conjugation of Ad to immunomodulatory cytokines (IL-4, IL-10,
and IFNÎł) did not detrimentally affect cytokine bioactivity
and enabled their sustained release. Our strategy of avidity-controlled
delivery of protein-based therapeutics is a promising approach for
the sustained local presentation of protein therapeutics and can be
applied to numerous biomedical applications
Computational Evaluation of HIV‑1 gp120 Conformations of Soluble Trimeric gp140 Structures as Targets for de Novo Docking of First- and Second-Generation Small-Molecule CD4 Mimics
Small-molecule CD4
mimics (SMCM’s) bind to the gp120 subunit
of the HIV-1 envelope glycoprotein (Env) and have been optimized to
block cell infection in vitro. The lack of the V1/2 and V3 loops and
the presence of the β2/3 and β20/21 strands (bridging
sheet) in the available structures of the monomeric gp120 core may
limit its applicability as a target for further synthetic optimization
of SMCM potency and/or breadth. Here, we employ a combination of binding-site
search, docking, estimation of protein–ligand interaction energy,
all-atom molecular dynamics, and ELISA-based CD4-binding competition
assays to create, characterize, and rationalize models of first- and
second-generation of SMCM’s bound to the distinct, trimeric
BG505 SOSIP.664 structures 4NCO and 4TVP containing V1/2 and V3 loops with no bridging sheet. We demonstrate
that the in silico neutralization of the highly conserved D368 is
necessary to obtain the correct orientation of SMCM in their binding
site when docking against the monomeric gp120 core. The computational
results correlate with IC<sub>50</sub>’s measured in CD4 binding
competition ELISA and with <i>K</i><sub>D</sub>’s
measured on gp120 core monomer. This supports the hypothesis that
the 4NCO trimeric
structure represents a viable target for further SMCM’s optimization
with advantages over both the 4TVP trimer and gp120 core monomer. Finally,
the docking protocol has been optimized to screen compounds that can
clearly interact with the highly conserved residue D368, increasing
the likelihood of future optimizations to arrive at SMCM’s
with a broader spectrum of activity
Peptide Triazole Inactivators of HIV‑1 Utilize a Conserved Two-Cavity Binding Site at the Junction of the Inner and Outer Domains of Env gp120
We
used coordinated mutagenesis, synthetic design, and flexible
docking to investigate the structural mechanism of Env gp120 encounter
by peptide triazole (PT) inactivators of HIV-1. Prior results demonstrated
that the PT class of inhibitors suppresses binding at both CD4 and
coreceptor sites on Env and triggers gp120 shedding, leading to cell-independent
irreversible virus inactivation. Despite these enticing anti-HIV-1
phenotypes, structural understanding of the PT–gp120 binding
mechanism has been incomplete. Here we found that PT engages two inhibitor
ring moieties at the junction between the inner and outer domains
of the gp120 protein. The results demonstrate how combined occupancy
of two gp120 cavities can coordinately suppress both receptor and
coreceptor binding and conformationally entrap the protein in a destabilized
state. The two-cavity model has common features with small molecule
gp120 inhibitor binding sites and provides a guide for further design
of peptidomimetic HIV-1 inactivators based on the PT pharmacophore
Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV‑1 by Peptide Triazole Thiols
We investigated the mode of action
underlying lytic inactivation
of HIV-1 virions by peptide triazole thiol (PTT), in particular the
relationship between gp120 disulfides and the C-terminal cysteine-SH
required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking
and PTTs with serially truncated linkers between pharmacophore isoleucine–ferrocenyltriazole-proline–tryptophan
and cysteine-SH were synthesized. PTT variants showed loss of lytic
activity but not binding and infection inhibition upon SH blockade.
A disproportionate loss of lysis activity vs binding and infection
inhibition was observed upon linker truncation. Molecular docking
of PTT onto gp120 argued that, with sufficient linker length, the
peptide SH could approach and disrupt several alternative gp120 disulfides.
Inhibition of lysis by gp120 mAb 2G12, which binds at the base of
the V3 loop, as well as disulfide mutational effects, argued that
PTT-induced disruption of the gp120 disulfide cluster at the base
of the V3 loop is an important step in lytic inactivation of HIV-1.
Further, PTT-induced lysis was enhanced after treating virus with
reducing agents dithiothreitol and tris (2-carboxyethyl)Âphosphine.
Overall, the results are consistent with the view that the binding
of PTT positions the peptide SH group to interfere with conserved
disulfides clustered proximal to the CD4 binding site in gp120, leading
to disulfide exchange in gp120 and possibly gp41, rearrangement of
the Env spike, and ultimately disruption of the viral membrane. The
dependence of lysis activity on thiol–disulfide interaction
may be related to intrinsic disulfide exchange susceptibility in gp120
that has been reported previously to play a role in HIV-1 cell infection
Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus–Cell Interface
To address the urgent
need for new agents to reduce the global
occurrence and spread of AIDS, we investigated the underlying hypothesis
that antagonists of the HIV-1 envelope (Env) gp120 protein and the
host-cell coreceptor (CoR) protein can be covalently joined into bifunctional
synergistic combinations with improved antiviral capabilities. A synthetic
protocol was established to covalently combine a CCR5 small-molecule
antagonist and a gp120 peptide triazole antagonist to form the bifunctional
chimera. Importantly, the chimeric inhibitor preserved the specific
targeting properties of the two separate chimera components and, at
the same time, exhibited low to subnanomolar potencies in inhibiting
cell infection by different pseudoviruses, which were substantially
greater than those of a noncovalent mixture of the individual components.
The results demonstrate that targeting the virus–cell interface
with a single molecule can result in improved potencies and also the
introduction of new phenotypes to the chimeric inhibitor, such as
the irreversible inactivation of HIV-1
Bifunctional Chimera That Coordinately Targets Human Immunodeficiency Virus 1 Envelope gp120 and the Host-Cell CCR5 Coreceptor at the Virus–Cell Interface
To address the urgent
need for new agents to reduce the global
occurrence and spread of AIDS, we investigated the underlying hypothesis
that antagonists of the HIV-1 envelope (Env) gp120 protein and the
host-cell coreceptor (CoR) protein can be covalently joined into bifunctional
synergistic combinations with improved antiviral capabilities. A synthetic
protocol was established to covalently combine a CCR5 small-molecule
antagonist and a gp120 peptide triazole antagonist to form the bifunctional
chimera. Importantly, the chimeric inhibitor preserved the specific
targeting properties of the two separate chimera components and, at
the same time, exhibited low to subnanomolar potencies in inhibiting
cell infection by different pseudoviruses, which were substantially
greater than those of a noncovalent mixture of the individual components.
The results demonstrate that targeting the virus–cell interface
with a single molecule can result in improved potencies and also the
introduction of new phenotypes to the chimeric inhibitor, such as
the irreversible inactivation of HIV-1