27 research outputs found
Improving the Resistance Profile of Hepatitis C NS3/4A Inhibitors: Dynamic Substrate Envelope Guided Design
Drug
resistance is a principal concern in the treatment of quickly
evolving diseases. The viral protease NS3/4A is a primary drug target
for the hepatitis C virus (HCV) and is known to evolve resistance
mutations in response to drug therapy. At the molecular level, drug
resistance reflects a subtle change in the balance of molecular recognition
by NS3/4A; the drug resistant protease variants are no longer effectively
inhibited by the competitive active site inhibitors but can still
process the natural substrates with enough efficiency for viral survival.
In previous works, we have developed the āsubstrate envelopeā
hypothesis, which posits that inhibitors should be less susceptible
to drug resistance if they better mimic the natural substrate molecular
recognition features. In this work, we perform molecular dynamics
simulations on four native substrates bound to NS3/4A and discover
a clearly conserved dynamic substrate envelope. We show that the most
severe drug resistance mutations in NS3/4A occur at residues that
are outside the substrate envelope. Comparative analysis of three
NS3/4A inhibitors reveals structural and dynamic characteristics of
inhibitors that could lead to resistance. We also suggest inhibitor
modifications to improve resistance profiles based on the dynamic
substrate envelope. This study provides a general framework for guiding
the development of novel inhibitors that will be more robust against
resistance by mimicking the static and dynamic binding characteristics
of natural substrates
HIV-1 Protease and Substrate Coevolution Validates the Substrate Envelope As the Substrate Recognition Pattern
Drug resistance of HIV-1 protease alters the balance
in the molecular
recognition events in favor of substrate processing versus inhibitor
binding. To develop robust inhibitors targeting ensembles of drug-resistant
variants, the code of this balance needs to be cracked. For this purpose,
the principles governing the substrate recognition are required to
be revealed. Previous crystallographic studies on the WT proteaseāsubstrate
complexes showed that the substrates have a conserved consensus volume
in the protease active site despite their low sequence homology. This
consensus volume is termed as the substrate envelope. The substrate
envelope was recently reevaluated by taking the substrate dynamics
into account, and the dynamic substrate envelope was reported to better
define the substrate specificity for HIV-1 protease. Drug resistance
occurs mostly through mutations in the protease, occasionally accompanied
by cleavage site mutations. In this study, three coevolved proteaseāsubstrate
complexes (<sup>AP2V</sup>NC-p1<sub>V82A</sub>, <sup>LP1ā²F</sup>p1-p6<sub>D30N/N88D</sub>, and <sup>SP3ā²N</sup>p1-p6<sub>D30N/N88D</sub>) were investigated for structural and dynamic properties by molecular
modeling and dynamics simulations. The results show the substrate
envelope is preserved by these cleavage site mutations in the presence
of drug-resistance mutations in the protease, if not enhanced. This
study on the conformational and mutational ensembles of proteaseāsubstrate
complexes validates the substrate envelope as the substrate recognition
motif for HIV-1 protease. The substrate envelope hypothesis allows
for the elucidation of possible drug resistance mutation patterns
in the polyprotein cleavage sites
Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease
Human immunodeficiency virus Type-1 (HIV-1) protease
is crucial
for viral maturation and infectivity. Studies of protease dynamics
suggest that the rearrangement of the hydrophobic core is essential
for enzyme activity. Many mutations in the hydrophobic core are also
associated with drug resistance and may modulate the core flexibility.
To test the role of flexibility in protease activity, pairs of cysteines
were introduced at the interfaces of flexible regions remote from
the active site. Disulfide bond formation was confirmed by crystal
structures and by alkylation of free cysteines and mass spectrometry.
Oxidized and reduced crystal structures of these variants show the
overall structure of the protease is retained. However, cross-linking
the cysteines led to drastic loss in enzyme activity, which was regained
upon reducing the disulfide cross-links. Molecular dynamics simulations
showed that altered dynamics propagated throughout the enzyme from
the engineered disulfide. Thus, altered flexibility within the hydrophobic
core can modulate HIV-1 protease activity, supporting the hypothesis
that drug resistant mutations distal from the active site can alter
the balance between substrate turnover and inhibitor binding by modulating
enzyme activity
Molecular and Dynamic Mechanism Underlying Drug Resistance in Genotype 3 Hepatitis C NS3/4A Protease
Hepatitis C virus
(HCV), affecting an estimated 150 million people
worldwide, is the leading cause of viral hepatitis, cirrhosis and
hepatocellular carcinoma. HCV is genetically diverse with six genotypes
(GTs) and multiple subtypes of different global distribution and prevalence.
Recent development of direct-acting antivirals against HCV including
NS3/4A protease inhibitors (PIs) has greatly improved treatment outcomes
for GT-1. However, all current PIs exhibit significantly lower potency
against GT-3. Lack of structural data on GT-3 protease has limited
our ability to understand PI failure in GT-3. In this study the molecular
basis for reduced potency of current inhibitors against GT-3 NS3/4A
protease is elucidated with structure determination, molecular dynamics
simulations and inhibition assays. A chimeric GT-1a3a NS3/4A protease
amenable to crystallization was engineered to recapitulate decreased
sensitivity of GT-3 protease to PIs. High-resolution crystal structures
of this GT-1a3a bound to 3 PIs, asunaprevir, danoprevir and vaniprevir,
had only subtle differences relative to GT-1 despite orders of magnitude
loss in affinity. In contrast, hydrogen-bonding interactions within
and with the protease active site and dynamic fluctuations of the
PIs were drastically altered. The correlation between loss of intermolecular
dynamics and inhibitor potency suggests a mechanism where polymorphisms
between genotypes (or selected mutations) in the drug target confer
resistance through altering the intermolecular dynamics of the proteināinhibitor
complex
Cooperative Effects of Drug-Resistance Mutations in the Flap Region of HIVā1 Protease
Understanding the interdependence of multiple mutations
in conferring
drug resistance is crucial to the development of novel and robust
inhibitors. As HIV-1 protease continues to adapt and evade inhibitors
while still maintaining the ability to specifically recognize and
efficiently cleave its substrates, the problem of drug resistance
has become more complicated. Under the selective pressure of therapy,
correlated mutations accumulate throughout the enzyme to compromise
inhibitor binding, but characterizing their energetic interdependency
is not straightforward. A particular drug resistant variant (L10I/G48V/I54V/V82A)
displays extreme entropyāenthalpy compensation relative to
wild-type enzyme but a similar variant (L10I/G48V/I54A/V82A) does
not. Individual mutations of sites in the flaps (residues 48 and 54)
of the enzyme reveal that the thermodynamic effects are not additive.
Rather, the thermodynamic profile of the variants is interdependent
on the cooperative effects exerted by a particular combination of
mutations simultaneously present
Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIVā1 Protease
Under the selective pressure of therapy,
HIV-1 protease mutants
resistant to inhibitors evolve to confer drug resistance. Such mutations
can impact both the dynamics and structures of the bound and unbound
forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1
protease with a combination of primary and secondary resistance mutations
(L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile
for darunavir (DRV) binding relative to the wild-type protease. We
elucidated the impact of these mutations on protein dynamics in the
DRV-bound state using molecular dynamics simulations and NMR relaxation
experiments. Both methods concur in that the conformational ensemble
and dynamics of protease are impacted by the drug resistance mutations
in Flap+ variant. Surprisingly this change in ensemble dynamics is
different from that observed in the unliganded form of the same variant
(Cai, Y. et al. <i>J. Chem. Theory Comput.</i> <b>2012</b>, <i>8</i>, 3452ā3462). Our comparative
analysis of both inhibitor-free and bound states presents a comprehensive
picture of the altered dynamics in drug-resistant mutant HIV-1 protease
and underlies the importance of incorporating dynamic analysis of
the whole system, including the unliganded state, into revealing drug
resistance mechanisms
Structural Analysis of the Active Site and DNA Binding of Human Cytidine Deaminase APOBEC3B
APOBEC3 (A3) proteins,
a family of human cytidine deaminases, protect
the host from endogenous retro-elements and exogenous viral infections
by introducing hypermutations. However, overexpressed A3s can modify
genomic DNA to promote tumorigenesis, especially A3B. Despite their
overall similarity, A3 proteins have distinct deamination activity.
Recently determined A3 structures have revealed the molecular determinants
of nucleotide specificity and DNA binding. However, for A3B, the structural
basis for regulation of deamination activity and the role of active
site loops in coordinating DNA had remained unknown. Using advanced
molecular modeling followed by experimental mutational analysis and
dynamics simulations, we investigated the molecular mechanism of DNA
binding by A3B-CTD. We modeled fully native A3B-DNA structure, and
we identified Arg211 in loop 1 as the gatekeeper coordinating DNA
and critical residue for nucleotide specificity. We also identified
a unique autoinhibited conformation in A3B-CTD that restricts access
and binding of DNA to the active site. Our results reveal the structural
basis for DNA binding and relatively lower catalytic activity of A3B
and provide opportunities for rational design of specific inhibitors
to benefit cancer therapeutics
Testing the Substrate-Envelope Hypothesis with Designed Pairs of Compounds
Acquired resistance to therapeutic
agents is a significant barrier to the development of clinically effective
treatments for diseases in which evolution occurs on clinical time
scales, frequently arising from target mutations. We previously reported
a general strategy to design effective inhibitors for rapidly mutating
enzyme targets, which we demonstrated for HIV-1 protease inhibition
[Altman et al. <i>J. Am. Chem. Soc.</i> 2008, <i>130</i>, 6099ā6113]. Specifically, we developed a computational inverse
design procedure with the added constraint that designed inhibitors
bind entirely inside the substrate envelope, a consensus volume occupied
by natural substrates. The rationale for the substrate-envelope constraint
is that it prevents designed inhibitors from making interactions beyond
those required by substrates and thus limits the availability of mutations
tolerated by substrates but not by designed inhibitors. The strategy
resulted in subnanomolar inhibitors that bind robustly across a clinically
derived panel of drug-resistant variants. To further test the substrate-envelope
hypothesis, here we have designed, synthesized, and assayed derivatives
of our original compounds that are larger and extend outside the substrate
envelope. Our designs resulted in pairs of compounds that are very
similar to one another, but one respects and one violates the substrate
envelope. The envelope-respecting inhibitor demonstrates robust binding
across a panel of drug-resistant protease variants, whereas the envelope-violating
one binds tightly to wild type but loses affinity to at least one
variant. This study provides strong support for the substrate-envelope
hypothesis as a design strategy for inhibitors that reduce susceptibility
to resistance mutations
Design, Synthesis, and Biological and Structural Evaluations of Novel HIV-1 Protease Inhibitors To Combat Drug Resistance
A series of new HIV-1 protease inhibitors (PIs) were
designed using
a general strategy that combines computational structure-based design
with substrate-envelope constraints. The PIs incorporate various alcohol-derived
P2 carbamates with acyclic and cyclic heteroatomic functionalities
into the (<i>R</i>)-hydroxyethylamine isostere. Most of
the new PIs show potent binding affinities against wild-type HIV-1
protease and three multidrug resistant (MDR) variants. In particular,
inhibitors containing the 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone,
and oxazolidinone moieties at P2 are the most potent with <i>K</i><sub>i</sub> values in the picomolar range. Several new
PIs exhibit nanomolar antiviral potencies against patient-derived
wild-type viruses from HIV-1 clades A, B, and C and two MDR variants.
Crystal structure analyses of four potent inhibitors revealed that
carbonyl groups of the new P2 moieties promote extensive hydrogen
bond interactions with the invariant Asp29 residue of the protease.
These structureāactivity relationship findings can be utilized
to design new PIs with enhanced enzyme inhibitory and antiviral potencies
Structural Adaptation of Darunavir Analogues against Primary Mutations in HIVā1 Protease
HIV-1 protease is one of the prime targets of agents used in antiretroviral
therapy against HIV. However, under selective pressure of protease
inhibitors, primary mutations at the active site weaken inhibitor
binding to confer resistance. Darunavir (DRV) is the most potent HIV-1
protease inhibitor in clinic; resistance is limited, as DRV fits well
within the substrate envelope. Nevertheless, resistance is observed
due to hydrophobic changes at residues including I50, V82, and I84
that line the S1/S1ā² pocket within the active site. Through
enzyme inhibition assays and a series of 12 crystal structures, we
interrogated susceptibility of DRV and two potent analogues to primary
S1ā² mutations. The analogues had modifications at the hydrophobic
P1ā² moiety compared to DRV to better occupy the unexploited
space in the S1ā² pocket where the primary mutations were located.
Considerable losses of potency were observed against protease variants
with I84V and I50V mutations for all three inhibitors. The crystal
structures revealed an unexpected conformational change in the flap
region of I50V protease bound to the analogue with the largest P1ā²
moiety, indicating interdependency between the S1ā² subsite
and the flap region. Collective analysis of proteaseāinhibitor
interactions in the crystal structures using principle component analysis
was able to distinguish inhibitor identity and relative potency solely
based on van der Waals contacts. Our results reveal the complexity
of the interplay between inhibitor P1ā² moiety and S1ā²
mutations and validate principle component analyses as a useful tool
for distinguishing resistance and inhibitor potency