Structure-Based Discovery and Development of Highly Potent Dihydroorotate Dehydrogenase Inhibitors for Malaria Chemoprevention.

Malaria remains a serious global health challenge, yet treatment and control programs are threatened by drug resistance. Dihydroorotate dehydrogenase (DHODH) was clinically validated as a target for treatment and prevention of malaria through human studies with DSM265, but currently no drugs against this target are in clinical use. We used structure-based computational tools including free energy perturbation (FEP+) to discover highly ligand efficient, potent, and selective pyrazole-based DHODH inhibitors through a scaffold hop from a pyrrole-based series.

Accurate Prediction of Protein Thermodynamic Stability Changes upon Residue Mutation using Free Energy Perturbation.

This work describes the application of a physics-based computational approach to predict the relative thermodynamic stability of protein variants, and evaluates the quantitative accuracy of those predictions compared to experimental data obtained from a diverse set of protein systems assayed at variable pH conditions. Physical stability is a key determinant of the clinical and commercial success of biological therapeutics, vaccines, diagnostics, enzymes and other protein-based products. Although experimental techniques for measuring the impact of amino acid residue mutation on the stability of proteins exist, they tend to be time consuming and costly, hence the need for accurate prediction methods.

Combination of HDX-MS and in silico modeling to study enzymatic reactivity and stereo-selectivity at different solvent conditions.

The higher-order structure of a protein defines its function, and protein structural dynamics are often essential for protein binding and enzyme catalysis. Methods for protein characterization in solution are continuously being developed to understand and explore protein conformational changes with regards to function and activity. The goal of this study was to survey the use of combining HDX-MS global conformational screening with in silico modeling and continuous labeling peptide-level HDX-MS as an approach to highlight regions of interest within an enzyme required for biocatalytic processes.

Design of an in vitro biocatalytic cascade for the manufacture of islatravir.

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates.

"Site and Mutation"-Specific Predictions Enable Minimal Directed Evolution Libraries.

Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes.

Identification of ortho-Substituted Benzoic Acid/Ester Derivatives via the Gas-Phase Neighboring Group Participation Effect in (+)-ESI High Resolution Mass Spectrometry.

Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical compounds and present a challenge in positional isomer identification by traditional tandem mass spectrometric analysis. A method is presented for exploiting the gas-phase neighboring group participation (NGP) effect to differentiate ortho-substituted benzoic acid/ester derivatives with high resolution mass spectrometry (HRMS). Significant water/alcohol loss (>30% abundance in MS spectra) was observed for ortho-substituted nucleophilic groups; these fragment peaks are not observable for the corresponding para and meta-substituted analogs.

Quantum-Mechanics Methodologies in Drug Discovery: Applications of Docking and Scoring in Lead Optimization.

The development and application of quantum mechanics (QM) methodologies in computer- aided drug design have flourished in the last 10 years. Despite the natural advantage of QM methods to predict binding affinities with a higher level of theory than those methods based on molecular mechanics (MM), there are only a few examples where diverse sets of protein-ligand targets have been evaluated simultaneously. In this work, we review recent advances in QM docking and scoring for those cases in which a systematic analysis has been performed.

Structural principles for computational and de novo design of 4Fe-4S metalloproteins.

Iron-sulfur centers in metalloproteins can access multiple oxidation states over a broad range of potentials, allowing them to participate in a variety of electron transfer reactions and serving as catalysts for high-energy redox processes. The nitrogenase FeMoCO cluster converts di-nitrogen to ammonia in an eight-electron transfer step. The 2(Fe4S4) containing bacterial ferredoxin is an evolutionarily ancient metalloprotein fold and is thought to be a primordial progenitor of extant oxidoreductases.

Energetic selection of topology in ferredoxins.

Models of early protein evolution posit the existence of short peptides that bound metals and ions and served as transporters, membranes or catalysts. The Cys-X-X-Cys-X-X-Cys heptapeptide located within bacterial ferredoxins, enclosing an Fe₄S₄ metal center, is an attractive candidate for such an early peptide. Ferredoxins are ancient proteins and the simple α+β fold is found alone or as a domain in larger proteins throughout all three kingdoms of life.

Computational design of thermostabilizing D-amino acid substitutions.

Judicious incorporation of D-amino acids in engineered proteins confers many advantages such as preventing degradation by endogenous proteases and promoting novel structures and functions not accessible to homochiral polypeptides. Glycine to D-alanine substitutions at the carboxy termini can stabilize α-helices by reducing conformational entropy. Beyond alanine, we propose additional side chain effects on the degree of stabilization conferred by D-amino acid substitutions.

Advantages and disadvantages of biodegradable platforms in drug eluting stents.

Coronary angioplasty with drug-eluting stent (DES) implantation is currently the most common stent procedure worldwide. Since the introduction of DES, coronary restenosis as well as the incidence of target vessel and target lesion revascularization have been significantly reduced. However, the incidence of very late stent thrombosis beyond the first year after stent deployment has more commonly been linked to DES than to bare-metal stent (BMS) implantation.

Effect of doxycycline on atherosclerosis: from bench to bedside.

Matrix metalloproteinases (MMPs) have a pivotal role in the natural history of atherosclerosis and its cardiovascular consequences. Non-selective MMP inhibition with doxycycline appears as a potential strategy to reduce the residual risk observed in patients already at intensive lipid lowering strategies. However, specific MMPs have different and even contradicting roles in the natural history of atherosclerosis, rendering broad spectrum MMP inhibition an important yet somewhat simplistic approach towards residual risk reduction in coronary atherosclerosis.

Copper-transfer mechanism from the human chaperone Atox1 to a metal-binding domain of Wilson disease protein.

The molecular details of how copper (Cu) is transferred from the human Cu chaperone Atox1 to metal-binding domains (MBDs) of P(1B)-type ATPases are still unclear. Here, we use a computational approach, employing quantum mechanics/molecular mechanics (QM/MM) methods, to shed light on the reaction mechanism [probable intermediates, Cu(I) coordination geometries, activation barriers, and energetics] of Cu(I) transfer from Atox1 to the fourth MBD of Wilson disease protein (WD4). Both Atox1 and WD4 have solvent-exposed metal-binding motifs with two Cys residues that coordinate Cu(I).

Interdomain interactions modulate collective dynamics of the metal-binding domains in the Wilson disease protein.

Wilson disease protein or ATP7B is a key player in human copper (Cu) homeostasis. Belonging to the P(1B) type subfamily of ATPases, its N-terminal region contains six soluble domains (WD1-WD6) connected by linkers that vary in length. These domains share a similar fold and bind Cu(I) in the conserved motif MCXXC.

Lysine-60 in copper chaperone Atox1 plays an essential role in adduct formation with a target Wilson disease domain.

The mechanism by which the human copper (Cu) chaperone Atox1 delivers Cu to metal-binding domains of Wilson disease (WD) protein for insertion into cuproenzymes is unclear. Using near-UV circular dichroism as a new tool to probe chaperone-target interactions, in combination with gel filtration and molecular dynamics simulations, we here demonstrate that Atox1 forms a stable Cu-dependent adduct with the fourth metal-binding domain of WD (WD4). Using point-mutated Atox1 variants, we show that the adduct forms in the absence of conserved residues M10 or T11 but K60 is essential for heterocomplex formation and Cu transfer.

Conformational dynamics of metal-binding domains in Wilson disease protein: molecular insights into selective copper transfer.

ATP7A/B are human P(1B)-type ATPases involved in cellular Cu homeostasis. The N-terminal parts of these multidomain proteins contain six metal-binding domains (MBDs) connected by linkers. The MBDs are similar in structure to each other and to the human copper chaperone Atox1, although their distinct roles in Cu transfer appear to vary.

Tuning of copper-loop flexibility in Bacillus subtilis CopZ copper chaperone: role of conserved residues.

Bacillus subtilis CopZ is a copper (Cu) chaperone that binds and delivers Cu to intracellular targets to maintain cellular Cu homeostasis. Like Cu chaperones from other organisms, including the human homologue Atox1, CopZ has the ferredoxin-like fold and binds Cu(I) via two Cys in a conserved M(11)X(12)C(13)X(14)X(15)C(16) motif located in a solvent-exposed loop. Here, we have performed extensive molecular dynamics simulations on strategic CopZ variants to reveal structural and dynamic roles of three residues near and in the Cu loop (i.

Differential roles of Met10, Thr11, and Lys60 in structural dynamics of human copper chaperone Atox1.

Atox1 is a human copper (Cu) chaperone with the ferredoxin-like fold that binds Cu(I) via two Cys residues in a M(10)X(11)C(12)X(13)X(14)C(15) motif located in a solvent-exposed loop. Here, we report molecular dynamics simulations that reveal the roles of Met10, Thr11, and Lys60 in Atox1 structural dynamics. Whereas Met10 is conserved in all Atox1 homologues, Thr11 and Lys60 are exchanged for Ser and Tyr in bacteria.

Stability and ATP binding of the nucleotide-binding domain of the Wilson disease protein: effect of the common H1069Q mutation.

Perturbation of the human copper-transporter Wilson disease protein (ATP7B) causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the nucleotide-binding subdomain (N-domain), including the most common mutation, H1069Q. To gain insight into the biophysical behavior of the N-domain under normal and disease conditions, we have characterized wild-type and H1069Q recombinant N-domains in vitro and in silico.

Structure and dynamics of Cu(I) binding in copper chaperones Atox1 and CopZ: a computer simulation study.

Copper chaperones deliver reduced copper (i.e., Cu(I)) to metal-binding domains of P-type ATPases in the cytoplasm of a range of organisms.

STAT5 transcriptional activity is impaired by LIF in a mammary epithelial cell line.

Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation.