Protein Electric Fields Enable Faster and Longer-Lasting Covalent Inhibition of ß-Lactamases.

The widespread design of covalent drugs has focused on crafting reactive groups of proper electrophilicity and positioning toward targeted amino-acid nucleophiles. We found that environmental electric fields projected onto a reactive chemical bond, an overlooked design element, play essential roles in the covalent inhibition of TEM-1 ß-lactamase by avibactam. Using the vibrational Stark effect, the magnitudes of the electric fields that are exerted by TEM active sites onto avibactam's reactive C═O were measured and demonstrate an electrostatic gating effect that promotes bond formation yet relatively suppresses the reverse dissociation. These results suggest new principles of covalent drug design and off-target site prediction. Unlike shape and electrostatic complementary which address binding constants, electrostatic catalysis drives reaction rates, essential for covalent inhibition, and deepens our understanding of chemical reactivity, selectivity, and stability in complex systems.

Molecular Mechanisms in the Selectivity of Nonsteroidal Anti-Inflammatory Drugs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) 1 and 2 with varying degrees of selectivity. A group of COX-2 selective inhibitors-coxibs-binds in a time-dependent manner through a three-step mechanism, utilizing a side pocket in the binding site. Coxibs have been extensively probed to identify the structural features regulating the slow tight-binding mechanism responsible for COX-2 selectivity. In this study, we further probe a structurally and kinetically diverse data set of COX inhibitors in COX-2 by molecular dynamics and free energy simulations. We find that the features regulating the high affinities associated with time-dependency in COX depend on the inhibitor kinetics. In particular, most time-dependent inhibitors share a common structural binding mechanism, involving an induced-fit rotation of the side-chain of Leu531 in the main binding pocket. The high affinities of two-step slow tight-binding inhibitors and some slow reversible inhibitors can thus be explained by the increased space in the main binding pocket after this rotation. Coxibs that belong to a separate class of slow tight-binding inhibitors benefit more from the displacement of the neighboring side-chain of Arg513, exclusive to the COX-2 side-pocket. This displacement further stabilizes the aforementioned rotation of Leu531 and can explain the selectivity of coxibs for COX-2.

Probing the Time Dependency of Cyclooxygenase-1 Inhibitors by Computer Simulations.

Time-dependent inhibition of the cyclooxygenases (COX) by a range of nonsteroidal anti-inflammatory drugs has been described since the first experimental assays of COX were performed. Slow tight-binding inhibitors of COX-1 bind in a two-step mechanism in which the EI → EI* transition is slow and practically irreversible. Since then, various properties of the inhibitors have been proposed to cause or affect the time dependency. Conformational changes in the enzyme have also been proposed to cause the time dependency, but no particular structural feature has been identified. Here, we investigated a series of inhibitors of COX-1 that are either time-independent or time-dependent using a combination of molecular dynamics simulations, binding free energy calculations, and potential of mean force calculations. We find that the time-dependent inhibitors stabilize a conformational change in the enzyme mainly identified by the rotation of a leucine side chain adjacent to the binding pocket. The induced conformation has been previously shown to be essential for the high binding affinities of tight-binding inhibitors in COX-1. The results of this work show that the structural features of the enzyme involved in both time-dependent and tight-binding inhibition are identical and further identify a structural mechanism responsible for the transition between the two enzyme-inhibitor complexes characteristic of slow tight-binding COX-1 inhibitors.

Origin of the Enigmatic Stepwise Tight-Binding Inhibition of Cyclooxygenase-1.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, inflammation, and some types of cancers. Their mechanism of action is the inhibition of isoforms 1 and 2 of the enzyme cyclooxygenase (COX-1 and COX-2, respectively). However, both nonselective and selective NSAIDs may have side effects that include gastric intestinal bleeding, peptic ulcer formation, kidney problems, and occurrences of myocardial infarction. The search for selective high-affinity COX inhibitors resulted in a number of compounds characterized by a slow, tight-binding inhibition that occurs in a two-step manner. It has been suggested that the final, only very slowly reversible, tight-binding event is the result of conformational changes in the enzyme. However, the nature of these conformational changes has remained elusive. Here we explore the structural determinants of the tight-binding phenomenon in COX-1 with molecular dynamics and free energy simulations. The calculations reveal how different classes of inhibitors affect the equilibrium between two conformational substates of the enzyme in distinctly different ways. The class of tight-binding inhibitors is found to exclusively stabilize an otherwise unfavorable enzyme conformation and bind significantly stronger to this state than to that normally observed in crystal structures. By also computing free energies of binding to the two enzyme conformations for 16 different NSAIDs, we identify an induced-fit mechanism and the key structural features associated with high-affinity tight binding. These results may facilitate the rational development of new COX inhibitors with improved selectivity profiles.

Toward an optimal docking and free energy calculation scheme in ligand design with application to COX-1 inhibitors.

Cyclooxygenase-1 (COX-1) is one of the main targets of most pain-relieving pharmaceuticals. Although the enzyme is well characterized, it is known to be a difficult target for automated molecular docking and scoring. We collected from the literature a structurally diverse set of 45 nonsteroidal anti-inflammatory drugs (NSAIDs) and COX-2-selective inhibitors (coxibs) with a wide range of binding affinities for COX-1. The binding of this data set to a homology model of human COX-1 was analyzed with different combinations of molecular docking algorithms, scoring functions, and the linear interaction energy (LIE) method for estimating binding affinities. It is found that the computational protocols for estimation of binding affinities are extremely sensitive to the initial orientations of the ligands in the binding pocket. To overcome this limitation, we propose a systematic exploration of docking poses using the LIE calculations as a postscoring function. This scheme yields predictions in excellent agreement with experiment, with a mean unsigned error of 0.9 kcal/mol for binding free energies and structures of high quality. A significant improvement of the results is also seen when averaging over experimental data from several independent measurements.

Simulation-guided engineering of split GFPs with efficient ß-strand photodissociation.

Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures.

The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of IRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10 500 drug-like substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors. A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons.