12 research outputs found
QM/MM simulations as an assay for carbapenemase activity in class A β-lactamases
Carbapenemases are distinguished from carbapenem-inhibited β-lactamases with a protocol involving QM/MM free energy simulations of acylâenzyme deacylation, requiring only the enzyme 3D structure as input.</p
QM/MM Simulations Reveal the Determinants of Carbapenemase Activity in Class A β-lactamases
[Image: see text] β-lactam antibiotic resistance in Gram-negative bacteria, primarily caused by β-lactamase enzymes that hydrolyze the β-lactam ring, has become a serious clinical problem. Carbapenems were formerly considered âlast resortâ antibiotics because they escaped breakdown by most β-lactamases, due to slow deacylation of the acyl-enzyme intermediate. However, an increasing number of Gram-negative bacteria now produce β-lactamases with carbapenemase activity: these efficiently hydrolyze the carbapenem β-lactam ring, severely limiting the treatment of some bacterial infections. Here, we use quantum mechanics/molecular mechanics (QM/MM) simulations of the deacylation reactions of acyl-enzyme complexes of eight β-lactamases of class A (the most widely distributed β-lactamase group) with the carbapenem meropenem to investigate differences between those inhibited by carbapenems (TEM-1, SHV-1, BlaC, and CTX-M-16) and those that hydrolyze them (SFC-1, KPC-2, NMC-A, and SME-1). QM/MM molecular dynamics simulations confirm the two enzyme groups to differ in the preferred acyl-enzyme orientation: carbapenem-inhibited enzymes favor hydrogen bonding of the carbapenem hydroxyethyl group to deacylating water (DW). QM/MM simulations of deacylation give activation free energies in good agreement with experimental hydrolysis rates, correctly distinguishing carbapenemases. For the carbapenem-inhibited enzymes, free energies for deacylation are significantly higher than for the carbapenemases, even when the hydroxyethyl group was restrained to prevent interaction with the DW. Analysis of these simulations, and additional simulations of mutant enzymes, shows how factors including the hydroxyethyl orientation, the active site volume, and architecture (conformations of Asn170 and Asn132; organization of the oxyanion hole; and the Cys69-Cys238 disulfide bond) collectively determine catalytic efficiency toward carbapenems
Using the fragment molecular orbital method to investigate agonistâorexin-2 receptor interactions
Using the fragment molecular orbital method to investigate agonist-orexin-2 receptor interactions
Abstract The understanding of binding interactions between any protein and a small molecule plays a key role in the rationalization of affinity and selectivity and is essential for an efficient structure-based drug discovery (SBDD) process. Clearly, to begin SBDD, a structure is needed, and although there has been fantastic progress in solving G-protein-coupled receptor (GPCR) crystal structures, the process remains quite slow and is not currently feasible for every GPCR or GPCR-ligand complex. This situation significantly limits the ability of X-ray crystallography to impact the drug discovery process for GPCR targets in 'real-time' and hence there is still a need for other practical and cost-efficient alternatives. We present here an approach that integrates our previously described hierarchical GPCR modelling protocol (HGMP) and the fragment molecular orbital (FMO) quantum mechanics (QM) method to explore the interactions and selectivity of the human orexin-2 receptor (OX 2 R) and its recently discovered nonpeptidic agonists. HGMP generates a 3D model of GPCR structures and its complexes with small molecules by applying a set of computational methods. FMO allows ab initio approaches to be applied to systems that conventional QM methods would find challenging. The key advantage of FMO is that it can reveal information on the individual contribution and chemical nature of each residue and water molecule to the ligand binding that normally would be difficult to detect without QM. We illustrate how the combination of both techniques provides a practical and efficient approach that can be used to analyse the existing structure-function relationships (SAR) and to drive forward SBDD in a real-world example for which there is no crystal structure of the complex available
The basis for carbapenem hydrolysis by class A β-lactamases: a combined investigation using crystallography and simulations
Carbapenems are the most potent β-lactam antibiotics and key drugs for treating infections by Gram-negative bacteria. In such organisms, β-lactam resistance arises principally from β-lactamase production. Although carbapenems escape the activity of most β-lactamases, due in the class A enzymes to slow deacylation of the covalent acylenzyme intermediate, carbapenem-hydrolyzing class A β-lactamases are now disseminating in clinically relevant bacteria. The reasons why carbapenems are substrates for these enzymes, but inhibit other class A β-lactamases, remain to be fully established. Here, we present crystal structures of the class A carbapenemase SFC-1 from Serratia fonticola and of complexes of its Ser70 Ala (Michaelis) and Glu166 Ala (acylenzyme) mutants with the carbapenem meropenem. These are the first crystal structures of carbapenem complexes of a class A carbapenemase. Our data reveal that, in the SFC-1 acylenzyme complex, the meropenem 6ι-1R-hydroxyethyl group interacts with Asn132, but not with the deacylating water molecule. Molecular dynamics simulations indicate that this mode of binding occurs in both the Michaelis and acylenzyme complexes of wild-type SFC-1. In carbapenem-inhibited class A β-lactamases, it is proposed that the deacylating water molecule is deactivated by interaction with the carbapenem 6ι-1R-hydroxyethyl substituent. Structural comparisons with such enzymes suggest that in SFC-1 subtle repositioning of key residues (Ser70, Ser130, Asn132 and Asn170) enlarges the active site, permitting rotation of the carbapenem 6ι-1R-hydroxyethyl group and abolishing this contact. Our data show that SFC-1, and by implication other such carbapenem-hydrolyzing enzymes, uses Asn132 to orient bound carbapenems for efficient deacylation and prevent their interaction with the deacylating water molecule.publishe
An Efficient Computational Assay for β-Lactam Antibiotic Breakdown by Class A β-Lactamases
Breakdown of β-lactam antibiotics by β-lactamases is one of the most common resistance mechanisms against these drugs. Here, we present a computationally efficient combined quantum mechanics/molecular mechanics simulation protocol for hydrolysis of the β-lactamase acylenzyme with meropenem, a carbapenem antibiotic. Starting from the previously published computational assay, we have successfully reduced both the conformational space and the sampling time, which leads to a more than 99% reduction on computer resources needed for this assay (less than 24 CPU hours per reaction). This simplified in silico protocol distinguishes accurately between class A β-lactamases that are able or unable to break down carbapenems. We further demonstrate that these protocols can be used to elucidate mechanistic details of β-lactam breakdown. The new efficient computational assaymay be used to predict carbapenem hydrolytic activity of new β-lactamase variants as they arise, and in identifying routes to development of new, more resilient β-lactam antibiotics or new generations of mechanism-based β-lactamase inhibitors.</div
The Basis for Carbapenem Hydrolysis by Class A βâLactamases: A Combined Investigation using Crystallography and Simulations
Carbapenems are the most potent β-lactam antibiotics
and
key drugs for treating infections by Gram-negative bacteria. In such
organisms, β-lactam resistance arises principally from β-lactamase
production. Although carbapenems escape the activity of most β-lactamases,
due in the class A enzymes to slow deacylation of the covalent acylenzyme
intermediate, carbapenem-hydrolyzing class A β-lactamases are
now disseminating in clinically relevant bacteria. The reasons why
carbapenems are substrates for these enzymes, but inhibit other class
A β-lactamases, remain to be fully established. Here, we present
crystal structures of the class A carbapenemase SFC-1 from Serratia fonticola and of complexes of its Ser70
Ala (Michaelis) and Glu166 Ala (acylenzyme) mutants with the carbapenem
meropenem. These are the first crystal structures of carbapenem complexes
of a class A carbapenemase. Our data reveal that, in the SFC-1 acylenzyme
complex, the meropenem 6Îą-1R-hydroxyethyl group interacts with
Asn132, but not with the deacylating water molecule. Molecular dynamics
simulations indicate that this mode of binding occurs in both the
Michaelis and acylenzyme complexes of wild-type SFC-1. In carbapenem-inhibited
class A β-lactamases, it is proposed that the deacylating water
molecule is deactivated by interaction with the carbapenem 6Îą-1R-hydroxyethyl
substituent. Structural comparisons with such enzymes suggest that
in SFC-1 subtle repositioning of key residues (Ser70, Ser130, Asn132
and Asn170) enlarges the active site, permitting rotation of the carbapenem
6Îą-1R-hydroxyethyl group and abolishing this contact. Our data
show that SFC-1, and by implication other such carbapenem-hydrolyzing
enzymes, uses Asn132 to orient bound carbapenems for efficient deacylation
and prevent their interaction with the deacylating water molecule
The Fragment Molecular Orbital Method Reveals New Insight into the Chemical Nature of GPCRâLigand Interactions
Our interpretation of ligandâprotein
interactions is often
informed by high-resolution structures, which represent the cornerstone
of structure-based drug design. However, visual inspection and molecular
mechanics approaches cannot explain the full complexity of molecular
interactions. Quantum Mechanics approaches are often too computationally
expensive, but one method, Fragment Molecular Orbital (FMO), offers
an excellent compromise and has the potential to reveal key interactions
that would otherwise be hard to detect. To illustrate this, we have
applied the FMO method to 18 Class A GPCRâligand crystal structures,
representing different branches of the GPCR genome. Our work reveals
key interactions that are often omitted from structure-based descriptions,
including hydrophobic interactions, nonclassical hydrogen bonds, and
the involvement of backbone atoms. This approach provides a more comprehensive
picture of receptorâligand interactions than is currently used
and should prove useful for evaluation of the chemical nature of ligand
binding and to support structure-based drug design
Nonempirical Energetic Analysis of Reactivity and Covalent Inhibition of Fatty Acid Amide Hydrolase
Fatty acid amide hydrolase (FAAH)
is a member of the amidase signature
family and is responsible for the hydrolytic deactivation of fatty
acid amide neuromodulators, such as anandamide. FAAH carries an unusual
catalytic triad consisting of Lys-Ser-Ser, which uniquely enables
the enzyme to cleave amides and esters at similar rates. The acylation
of 9<i>Z</i>-octadecenamide (oleamide, a FAAH reference
substrate) has been widely investigated by computational methods,
and those have shown that conformational fluctuations of the active
site affect the reaction barrier. Empirical descriptors have been
devised to provide a possible mechanistic explanation for such conformational
effects, but a first-principles understanding is still missing. A
comparison of FAAH acylation with a reference reaction in water suggests
that transition-state stabilization is crucial for catalysis because
the activation energy barrier falls by 6 kcal/mol in the presence
of the active site. With this in mind, we have analyzed the enzymatic
reaction using the differential transition-state stabilization (DTSS)
approach to determine key active-site residues for lowering the barrier.
We examined several QM/MM structures at the MP2 level of theory and
analyzed catalytic effects with a variationâperturbation partitioning
of the interaction energy into electrostatic multipole and penetration,
exchange, delocalization, and correlation terms. Three residues â
Thr236, Ser218, and one water molecule â appear to be essential
for the stabilization of the transition state, a conclusion that is
also reflected by catalytic fields and agrees with site-directed mutagenesis
data. An analogous analysis for URB524, URB618, and URB694 (three
potent representatives of covalent, carbamate-based FAAH inhibitors)
confirms the importance of the residues involved in oleamide acylation,
providing insight for future inhibitor design
Fragment Molecular Orbital Method Applied to Lead Optimization of Novel Interleukinâ2 Inducible TâCell Kinase (ITK) Inhibitors
Inhibition
of inducible T-cell kinase (ITK), a nonreceptor tyrosine
kinase, may represent a novel treatment for allergic asthma. In our
previous reports, we described the discovery of sulfonylpyridine (SAP),
benzothiazole (BZT), indazole (IND), and tetrahydroindazole (THI)
series as novel ITK inhibitors and how computational tools such as
dihedral scans and docking were used to support this process. X-ray
crystallography and modeling were applied to provide essential insight
into ITKâligand interactions. However, âvisual inspectionâ
traditionally used for the rationalization of proteinâligand
affinity cannot always explain the full complexity of the molecular
interactions. The fragment molecular orbital (FMO) quantum-mechanical
(QM) method provides a complete list of the interactions formed between
the ligand and protein that are often omitted from traditional structure-based
descriptions. FMO methodology was successfully used as part of a rational
structure-based drug design effort to improve the ITK potency of high-throughput
screening hits, ultimately delivering ligands with potency in the
subnanomolar range