James Andrew McCammon

Maximizing clinical benefits of therapeutics while minimizing adverse effects is a central challenge in drug development. For Parkinson's disease (PD), l-3,4-dihydroxyphenylalanine (l-dopa) is the most effective treatment available, but chronic use is associated with periods of reduced efficacy (motor fluctuations) and the debilitating on-target side effect of l-dopa-induced dyskinesia. To disentangle the molecular mechanisms underlying l-dopa's antiparkinsonian effects versus its dyskinetic effects, we analyzed transcriptomic data from the striata of mice exposed to different l-dopa doses, identifying distinct gene expression signatures associated with l-dopa's effect on parkinsonian motor symptoms and dyskinesia. By comparing these gene expression signatures with a large dataset of in vitro gene expression profiles of drug perturbations, we identified SB-0107, a positive allosteric modulator of protein kinase A type II with previous clinical experience that boosts l-dopa's benefits while reducing dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned macaque model of PD. Deuterated analogs, including SB-0110, were developed that improved both pharmacokinetics and pharmacological effects. This dual effect, established safety profile of the parent compound, and improved properties of the deuterated analog support further development of this compound as an adjunct treatment to l-dopa therapy for PD.

In the Big Data era, a change of paradigm in the use of molecular dynamics is required. Trajectories should be stored under FAIR (findable, accessible, interoperable and reusable) requirements to favor its reuse by the community under an open science paradigm.

Group VIA calcium-independent phospholipase A ₂ (iPLA ₂ ) is a member of the PLA ₂ superfamily that exhibits calcium-independent activity in contrast to the other two major types, secreted phospholipase A ₂ (sPLA ₂ ) and cytosolic phospholipase A ₂ (cPLA ₂ ), which both require calcium for their enzymatic activity. Adenosine triphosphate (ATP) has been reported to allosterically activate iPLA ₂ , and this has now been verified with a lipidomics-based mixed-micelle assay, but its mechanism of action has been unknown. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to identify ATP interaction peptide regions located within the ankyrin repeat domain at which ATP interacts. Molecular dynamics simulations revealed the mechanism by which ATP binds to its site and the main residues that interact. Site-directed mutagenesis was used to verify the importance of these residues in the role of ATP in regulating iPLA ₂ activity. Importantly, calcium was found to abolish the enhancing regulatory function of ATP and to promote the inhibitory activity by calmodulin. Given previous evidence that calcium does not bind directly to iPLA ₂ , its effect appears to be indirect via association with ATP and/or calmodulin. Using HDX-MS, we found that calmodulin interacts with the N terminus peptide region of iPLA ₂ consisting of residues 20 to 28. These two regulatory iPLA ₂ sites open the road to the development of potential targets for therapeutic intervention.

The roles of computing in structure-based drug discovery are considered, from early studies based on some of the first experimental structures of enzyme-inhibitor complexes, through the use of advanced molecular dynamics simulations and machine learning methods. This perspective aims to explore the history, current trends, and future directions of these methodologies.

We present the NetSci program-an open-source scientific software package designed for estimating mutual information (MI) between data sets using GPU acceleration and a k-nearest-neighbor algorithm. This approach significantly enhances calculation speed, achieving improvements of several orders of magnitude over traditional CPU-based methods, with data set size limits dictated only by available hardware. To validate NetSci, we accurately compute MI for an analytically verifiable two-dimensional Gaussian distribution and replicate the generalized correlation (GC) analysis previously conducted on the B1 domain of protein G. We also apply NetSci to molecular dynamics simulations of the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA) pump, exploring the allosteric mechanisms and pathways influenced by ATP and 2'-deoxy-ATP (dATP) binding. Our analysis reveals distinct allosteric effects induced by ATP compared to dATP, with predicted information pathways from the bound nucleotide to the calcium-binding domain differing based on the nucleotide involved. NetSci proves to be a valuable tool for estimating MI and GC in various data sets and is particularly effective for analyzing intraprotein communication and information transfer.

2’-deoxy-ATP (dATP) improves cardiac function by increasing the rate of crossbridge cycling and Ca 2 + transient decay. However, the mechanisms of these effects and how therapeutic responses to dATP are achieved when dATP is only a small fraction of the total ATP pool remain poorly understood. Here, we used a multiscale computational modeling approach to analyze the mechanisms by which dATP improves ventricular function. We integrated atomistic simulations of prepowerstroke myosin and actomyosin association, filament-scale Markov state modeling of sarcomere mechanics, cell-scale analysis of myocyte Ca 2 + dynamics and contraction, organ-scale modeling of biventricular mechanoenergetics, and systems level modeling of circulatory dynamics. Molecular and Brownian dynamics simulations showed that dATP increases the actomyosin association rate by 1.9 fold. Markov state models predicted that dATP increases the pool of myosin heads available for crossbridge cycling, increasing steady-state force development at low dATP fractions by 1.3 fold due to mechanosensing and nearest-neighbor cooperativity. This was found to be the dominant mechanism by which small amounts of dATP can improve contractile function at myofilament to organ scales. Together with faster myocyte Ca 2 + handling, this led to improved ventricular contractility, especially in a failing heart model in which dATP increased ejection fraction by 16% and the energy efficiency of cardiac contraction by 1%. This work represents a complete multiscale model analysis of a small molecule myosin modulator from single molecule to organ system biophysics and elucidates how the molecular mechanisms of dATP may improve cardiovascular function in heart failure with reduced ejection fraction.

Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k_cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K_ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first determined in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis (Mtb). Most previous drug discovery efforts toward cytochrome bd have involved analogues of the canonical substrate quinone, known as Aurachin D. Here, we report six new cytochrome bd inhibitor scaffolds determined from a computational screen and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization toward Mtb therapeutics.

We present the Netsci program - an open-source scientific software package that leverages GPU acceleration and a k-nearest-neighbor algorithm in order to estimate the mutual information (MI) between data in a set. The GPU acceleration presented here, as an improvement upon existing estimators, enables calculation speeds several orders of magnitude faster than CPU-based implementations, all with dataset size limits determined only by the available hardware. To demonstrate the validity and usefulness of Netsci, we show that the MI is correctly computed for the analytically-verifiable two-dimensional Gaussian distribution, and we also reproduce the generalized correlation (GC) analysis performed in an earlier study on the B1 domain of protein G. In addition, we apply Netsci to the analysis of molecular dynamics simulations of the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA) pump. Specifically, we use Netsci to understand the allosteric mechanisms and pathways of SERCA, and compare the differential effects of the binding of two nucleotides, ATP and 2'-deoxy-ATP (dATP). We determine that ATP binding to SERCA, compared to dATP, induces differential allosteric effects. The most likely information pathways from the bound nucleotide to the calcium binding domain are also predicted using our MI estimator in combination with network analysis tools on the SERCA pump, which differs based on the bound nucleotide. Netsci is shown to be a useful program for the estimation of MI and GC within general datasets, and for the analysis of intraprotein communication and information transfer, in particular.

Plants respond to increased CO₂ concentrations through stomatal closure, which can contribute to increased water use efficiency. Grasses display faster stomatal responses than eudicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO₂ signal transduction mutants in grasses has yet to be reported. The grass model Brachypodium distachyon is closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO₂ control mechanisms of stomatal movements in grasses, we developed an unbiased forward genetic screen with an EMS-mutagenized Brachypodium distachyon M5 generation population using infrared imaging to identify plants with altered leaf temperatures at elevated CO₂. Among isolated mutants, a “chill1” mutant exhibited cooler leaf temperatures than wildtype Bd21-3 parent control plants after exposure to increased [CO₂]. chill1 plants showed strongly impaired high CO₂-induced stomatal closure despite retaining a robust abscisic acid-induced stomatal closing response. Through bulked segregant whole-genome-sequencing analyses followed by analyses of further backcrossed F4 generation plants and generation and characterization of sodium-azide and CRISPR-cas9 mutants, chill1 was mapped to a protein kinase, Mitogen-Activated Protein Kinase 5 (BdMPK5). The chill1 mutation impaired BdMPK5 protein-mediated CO₂/HCO₃^- sensing together with the High Temperature 1 (HT1) Raf-like kinase in vitro. Furthermore, AlphaFold2-directed structural modeling predicted that the identified BdMPK5-D90N chill1 mutant residue is located at the interface of BdMPK5 with the BdHT1 Raf-like kinase. BdMPK5 is a key signaling component that mediates CO₂-induced stomatal movements and is proposed to function as a component of the primary CO₂ sensor in grasses.