Martini 3 Force Field Parameters for Protein Lipidation Post-Translational Modifications.

Protein lipidations are vital co/post-translational modifications that tether lipid tails to specific protein amino acids, allowing them to anchor to biological membranes, switch their subcellular localization, and modulate association with other proteins. Such lipidations are thus crucial for multiple biological processes including signal transduction, protein trafficking, and membrane localization and are implicated in various diseases as well. Examples of lipid-anchored proteins include the Ras family of proteins that undergo farnesylation; actin and gelsolin that are myristoylated; phospholipase D that is palmitoylated; glycosylphosphatidylinositol-anchored proteins; and others. Here, we develop parameters for cysteine-targeting farnesylation, geranylgeranylation, and palmitoylation, as well as glycine-targeting myristoylation for the latest version of the Martini 3 coarse-grained force field. The parameters are developed using the CHARMM36m all-atom force field parameters as reference. The behavior of the coarse-grained models is consistent with that of the all-atom force field for all lipidations and reproduces key dynamical and structural features of lipid-anchored peptides, such as the solvent-accessible surface area, bilayer penetration depth, and representative conformations of the anchors. The parameters are also validated in simulations of the lipid-anchored peripheral membrane proteins Rheb and Arf1, after comparison with independent all-atom simulations. The parameters, along with mapping schemes for the popular martinize2 tool, are available for download at 10.5281/zenodo.7849262 and also as supporting information.

Membrane Composition and Raf[CRD]-Membrane Attachment Are Driving Forces for K-Ras4B Dimer Stability.

Ras proteins are membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the properties of Ras in terms of dimerization/clustering on the cell membrane. To understand the effects of anionic lipids on key spatiotemporal properties of dimeric K-Ras4B, we perform all-atom molecular dynamics simulations of the dimer K-Ras4B in the presence and absence of Raf[RBD/CRD] effectors on two model anionic lipid membranes: one containing 78% mol DOPC, 20% mol DOPS, and 2% mol PIP2 and another one with enhanced concentration of anionic lipids containing 50% mol DOPC, 40% mol DOPS, and 10% mol PIP2. Analysis of our results unveils the orientational space of dimeric K-Ras4B and shows that the stability of the dimer is enhanced on the membrane containing a high concentration of anionic lipids in the absence of Raf effectors. This enhanced stability is also observed in the presence of Raf[RBD/CRD] effectors although it is not influenced by the concentration of anionic lipids in the membrane, but rather on the ability of Raf[CRD] to anchor to the membrane. We generate dominant K-Ras4B conformations by Markov state modeling and yield the population of states according to the K-Ras4B orientation on the membrane. For the membrane containing anionic lipids, we observe correlations between the diffusion of K-Ras4B and PIP2 and anchoring of anionic lipids to the Raf[CRD] domain. We conclude that the presence of effectors with the Raf[CRD] domain anchoring on the membrane as well as the membrane composition both influence the conformational stability of the K-Ras4B dimer, enabling the preservation of crucial interface interactions.

Use of Intramolecular 1,5-Sulfur-Oxygen and 1,5-Sulfur-Halogen Interactions in the Design of N-Methyl-5-aryl-N-(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,4-thiadiazol-2-amine SMN2 Splicing Modulators.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by low levels of functional survival motor neuron protein (SMN) resulting from a deletion or loss of function mutation of the survival motor neuron 1 (SMN1) gene. Branaplam (1) elevates levels of full-length SMN protein in vivo by modulating the splicing of the related gene SMN2 to enhance the exon-7 inclusion and increase levels of the SMN. The intramolecular hydrogen bond present in the 2-hydroxyphenyl pyridazine core of 1 enforces a planar conformation of the biaryl system and is critical for the compound activity. Scaffold morphing revealed that the pyridazine could be replaced by a 1,3,4-thiadiazole, which provided additional opportunities for a conformational constraint of the biaryl through intramolecular 1,5-sulfur-oxygen (S···O) or 1,5-sulfur-halogen (S···X) noncovalent interactions. Compound 26, which incorporates a 2-fluorophenyl thiadiazole motif, demonstrated a greater than 50% increase in production of full-length SMN protein in a mouse model of SMA.

Evaluating the Efficiency of the Martini Force Field to Study Protein Dimerization in Aqueous and Membrane Environments.

Protein-protein complex assembly is one of the major drivers of biological response. Understanding the mechanisms of protein oligomerization/dimerization would allow one to elucidate how these complexes participate in biological activities and could ultimately lead to new approaches in designing novel therapeutic agents. However, determining the exact association pathways and structures of such complexes remains a challenge. Here, we use parallel tempering metadynamics simulations in the well-tempered ensemble to evaluate the performance of Martini 2.2P and Martini open-beta 3 (Martini 3) force fields in reproducing the structure and energetics of the dimerization process of membrane proteins and proteins in an aqueous solution in reasonable accuracy and throughput. We find that Martini 2.2P systematically overestimates the free energy of association by estimating large barriers in distinct areas, which likely leads to overaggregation when multiple monomers are present. In comparison, the less viscous Martini 3 results in a systematic underestimation of the free energy of association for proteins in solution, while it performs well in describing the association of membrane proteins. In all cases, the near-native dimer complexes are identified as minima in the free energy surface albeit not always as the lowest minima. In the case of Martini 3, we find that the spurious supramolecular protein aggregation present in Martini 2.2P multimer simulations is alleviated and thus this force field may be more suitable for the study of protein oligomerization. We propose that the use of enhanced sampling simulations with a refined coarse-grained force field and appropriately defined collective variables is a robust approach for studying the protein dimerization process, although one should be cautious of the ranking of energy minima.

Evolution of Novartis' Small Molecule Screening Deck Design.

This article summarizes the evolution of the screening deck at the Novartis Institutes for BioMedical Research (NIBR). Historically, the screening deck was an assembly of all available compounds. In 2015, we designed a first deck to facilitate access to diverse subsets with optimized properties. We allocated the compounds as plated subsets on a 2D grid with property based ranking in one dimension and increasing structural redundancy in the other. The learnings from the 2015 screening deck were applied to the design of a next generation in 2019. We found that using traditional leadlikeness criteria (mainly MW, clogP) reduces the hit rates of attractive chemical starting points in subset screening. Consequently, the 2019 deck relies on solubility and permeability to select preferred compounds. The 2019 design also uses NIBR's experimental assay data and inferred biological activity profiles in addition to structural diversity to define redundancy across the compound sets.

PathwayMap: Molecular Pathway Association with Self-Normalizing Neural Networks.

Drug discovery suffers from high attrition because compounds initially deemed as promising can later show ineffectiveness or toxicity resulting from a poor understanding of their activity profile. In this work, we describe a deep self-normalizing neural network model for the prediction of molecular pathway association and evaluate its performance, showing an AUC ranging from 0.69 to 0.91 on a set of compounds extracted from ChEMBL and from 0.81 to 0.83 on an external data set provided by Novartis. We finally discuss the applicability of the proposed model in the domain of lead discovery. A usable application is available via PlayMolecule.org .

Discovery of dual GyrB/ParE inhibitors active against Gram-negative bacteria.

Even though many GyrB and ParE inhibitors have been reported in the literature, few possess activity against Gram-negative bacteria. This is primarily due to limited permeability across Gram-negative bacterial membrane as well as bacterial efflux mechanisms. Permeability of compounds across Gram-negative bacterial membranes depends on many factors including physicochemical properties of the inhibitors. Herein, we show the optimization of pyridylureas leading to compounds with potent activity against Gram-negative bacterial species such as P.aeruginosa, E.coli and A.baumannii.

Structure-Based Design of Selective Noncovalent CDK12 Inhibitors.

Cyclin-dependent kinase (CDK) 12 knockdown via siRNA decreases the transcription of DNA-damage-response genes and sensitizes BRCA wild-type cells to poly(ADP-ribose) polymerase (PARP) inhibition. To recapitulate this effect with a small molecule, we sought a potent, selective CDK12 inhibitor. Crystal structures and modeling informed hybridization between dinaciclib and SR-3029, resulting in lead compound 5 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-ethyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Further structure-guided optimization delivered a series of selective CDK12 inhibitors, including compound 7 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-isopropyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Profiling of this compound across CDK9, 7, 2, and 1 at high ATP concentration, single-point kinase panel screening against 352 targets at 0.1 µm, and proteomics via kinase affinity matrix technology demonstrated the selectivity. This series of compounds inhibits phosphorylation of Ser2 on the C-terminal repeat domain of RNA polymerase II, consistent with CDK12 inhibition. These selective compounds were also acutely toxic to OV90 as well as THP1 cells.

ESKAPEing the labyrinth of antibacterial discovery.

Antimicrobial drug resistance is a growing threat to global public health. Multidrug resistance among the 'ESKAPE' organisms - encompassing Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. - is of particular concern because they are responsible for many serious infections in hospitals. Although some promising agents are in the pipeline, there is an urgent need for new antibiotic scaffolds. However, antibacterial researchers have struggled to identify new small molecules with meaningful cellular activity, especially those effective against multidrug-resistant Gram-negative pathogens. This difficulty ultimately stems from an incomplete understanding of efflux systems and compound permeation through bacterial membranes. This Opinion article describes findings from target-based and phenotypic screening efforts carried out at AstraZeneca over the past decade, discusses some of the subsequent chemistry challenges and concludes with a description of new approaches comprising a combination of computational modelling and advanced biological tools which may pave the way towards the discovery of new antibacterial agents.

Toward the rational design of carbapenem uptake in Pseudomonas aeruginosa.

Understanding how compound penetration occurs across the complex cell walls of Gram-negative bacteria is one of the greatest challenges in discovering new drugs to treat the infections they cause. A combination of next-generation transposon sequencing, computational metadynamics simulations (CMDS), and medicinal chemistry was used to define genetic and structural elements involved in facilitated carbapenem entry into Pseudomonas aeruginosa. Here we show for the first time that these compounds are taken up not only by the major outer membrane channel OccD1 (also called OprD or PA0958) but also by a closely related channel OccD3 (OpdP or PA4501). Transport-mediating molecular interactions predicted by CMDS for these channels were first confirmed genetically, then used to guide the design of carbapenem analogs with altered uptake properties. These results bring us closer to the rational design of channel transmissibility and may ultimately lead to improved permeability of compounds across bacterial outer membranes.

A New-Class Antibacterial-Almost. Lessons in Drug Discovery and Development: A Critical Analysis of More than 50 Years of Effort toward ATPase Inhibitors of DNA Gyrase and Topoisomerase IV.

The introduction into clinical practice of an ATPase inhibitor of bacterial DNA gyrase and topoisomerase IV (topo IV) would represent a new-class agent for the treatment of resistant bacterial infections. Novobiocin, the only historical member of this class, established the clinical proof of concept for this novel mechanism during the late 1950s, but its use declined rapidly and it was eventually withdrawn from the market. Despite significant and prolonged effort across the biopharmaceutical industry to develop other agents of this class, novobiocin remains the only ATPase inhibitor of gyrase and topo IV ever to progress beyond Phase I. In this review, we analyze the historical attempts to discover and develop agents within this class and highlight factors that might have hindered those efforts. Within the last 15 years, however, our technical understanding of the molecular details of the inhibition of the gyrase and topo IV ATPases, the factors governing resistance development to such inhibitors, and our knowledge of the physical properties required for robust clinical drug candidates have all matured to the point wherein the industry may now address this mechanism of action with greater confidence. The antibacterial spectrum within this class has recently been extended to begin to include serious Gram negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. In spite of this recent technical progress, adverse economics associated with antibacterial R&D over the last 20 years has diminished industry's ability to commit the resources and perseverance needed to bring new-class agents to launch. Consequently, a number of recent efforts in the ATPase class have been derailed by organizational rather than scientific factors. Nevertheless, within this context we discuss the unique opportunity for the development of ATPase inhibitors of gyrase and topo IV as new-class antibacterial agents with broad spectrum potential.

Optimization of pyrrolamide topoisomerase II inhibitors toward identification of an antibacterial clinical candidate (AZD5099).

AZD5099 (compound 63) is an antibacterial agent that entered phase 1 clinical trials targeting infections caused by Gram-positive and fastidious Gram-negative bacteria. It was derived from previously reported pyrrolamide antibacterials and a fragment-based approach targeting the ATP binding site of bacterial type II topoisomerases. The program described herein varied a 3-piperidine substituent and incorporated 4-thiazole substituents that form a seven-membered ring intramolecular hydrogen bond with a 5-position carboxylic acid. Improved antibacterial activity and lower in vivo clearances were achieved. The lower clearances were attributed, in part, to reduced recognition by the multidrug resistant transporter Mrp2. Compound 63 showed notable efficacy in a mouse neutropenic Staphylococcus aureus infection model. Resistance frequency versus the drug was low, and reports of clinical resistance due to alteration of the target are few. Hence, 63 could offer a novel treatment for serious issues of resistance to currently used antibacterials.

Fragment-to-hit-to-lead discovery of a novel pyridylurea scaffold of ATP competitive dual targeting type II topoisomerase inhibiting antibacterial agents.

The discovery and optimization of a new class of bacterial topoisomerase (DNA gyrase and topoisomerase IV) inhibitors binding in the ATP domain are described. A fragment molecule, 1-ethyl-3-(2-pyridyl)urea, provided sufficiently potent enzyme inhibition (32 µM) to prompt further analogue work. Acids and acid isosteres were incorporated at the 5-pyridyl position of this fragment, bridging to a key asparagine residue, improving enzyme inhibition, and leading to measurable antibacterial activity. A CF3-thiazole substituent at the 4-pyridyl position improved inhibitory potency due to a favorable lipophilic interaction. Promising antibacterial activity was seen versus the Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae and the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis . Precursor metabolite incorporation and mutant analysis studies support the mode-of-action, blockage of DNA synthesis by dual target topoisomerase inhibition. Compound 35 was efficacious in a mouse S. aureus disease model, where a 4.5-log reduction in colony forming units versus control was demonstrated.

Novel topoisomerase inhibitors: microbiological characterisation and in vivo efficacy of pyrimidines.

Pyrimidine compounds were identified as inhibitors of DNA topoisomerase IV through high-throughput screening. This study was designed to exemplify the in vitro activity of the pyrimidines against Gram-positive and Gram-negative microorganisms, to reveal the mode of action of these compounds and to demonstrate their in vivo efficacy. Frequencies of resistance to pyrimidines among Staphylococcus aureus and Streptococcus pneumoniae were <10(-10) at four times their minimum inhibitory concentrations (MICs). These compounds exhibited a dual mode of action through inhibition of the ParE subunit of DNA topoisomerase IV as well as the GyrB subunit of DNA gyrase, a homologue of DNA topoisomerase IV. Pyrimidines were shown to have MIC(90) values (MIC that inhibited 90% of the strains tested) of ≤2 mg/L against Gram-positive pathogens, including meticillin-resistant S. aureus, quinolone- and meticillin-resistant S. aureus, vancomycin-resistant enterococci, penicillin-non-susceptible S. pneumoniae and Streptococcus pyogenes, and MIC(90) values of 2- to >16 mg/L and ≤0.5 mg/L against the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis, respectively. The pyrimidines were bactericidal and exhibited a ca. 1000-fold reduction of the bacterial counts at 300 mg/kg in a S. pneumoniae lung infection model. The microbiological properties and in vivo efficacy of pyrimidines underscore their potential as candidates for the treatment of soft-tissue infections and hospital-acquired pneumonia.

Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and Topoisomerase IV.

We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 µM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.

Antimicrobial Activity of Adenine-Based Inhibitors of NAD(+)-Dependent DNA Ligase.

The relationship between enzyme inhibition and antimicrobial potency of adenine-based NAD(+)-dependent DNA ligase (LigA) inhibitors was investigated using a strain of the Gram-negative pathogen Haemophilus influenzae lacking its major AcrAB-TolC efflux pump and the Gram-positive pathogen Streptococcus pneumoniae. To this end, biochemical inhibitors not mediating their antibacterial mode of action (MOA) via LigA were removed from the analysis. In doing so, a significant number of compounds were identified that acted via inhibition of LigA in S. pneumoniae but not in H. influenzae, despite being inhibitors of both isozymes. Deviations from the line correlating antimicrobial and biochemical potencies of LigA inhibitors with the correct MOA were observed for both species. These deviations, usually corresponding to higher MIC/IC50 ratios, were attributed to varying compound permeance into the cell.

Evaluation of pKa estimation methods on 211 druglike compounds.

The pK(a) values of 211 discovery (druglike) compounds were determined experimentally using capillary electrophoresis coupled with ultraviolet spectroscopy and a novel fitting algorithm. These values were compared to those predicted by five different commercially available pK(a) estimation packages: ACDLabs/pK(a), Marvin (ChemAxon), MoKa (Molecular Discovery), Epik (Schrodinger), and Pipeline Pilot (Accelrys). Even though the topological method MoKa was noticeably faster than ACD, the accuracy of those two methods and Marvin was statistically indistinguishable, with a root-mean-squared error of about 1 pK(a) unit compared to experiment. Pipeline Pilot and EpiK both produced pK(a) estimates in significantly worse agreement with the experiment. Interestingly, on a number of compounds, the predictions due to ACD v12 were in poorer agreement with the experiment than ACD v10. Microscopic and "apparent" pK(a) predictions were also compared using ACD v10. Microscopic pK(a)s gave significantly worse agreement with the experiment than the "apparent" values. In all cases, the errors appeared to be randomly distributed across chemical series.

CAUTION: popular "benchmark" data sets do not distinguish the merits of 3D QSAR methods.

The quality of 3D QSAR models obtained using extremely simple descriptors was examined for nine popular data sets, including the well-known set of 31 steroids, which for 20 years has been the standard for benchmarking 3D QSAR methods. The atomic numbers of atoms coinciding with vertices of the molecular alignment as well as binary descriptors indicating the occupancy of those vertices were compared to models obtained using SAMFA descriptors, which have been shown to yield models statistically indistinguishable from CoMFA. For most data sets, only a minor loss in model performance was observed, even for the occupancy descriptors, where all chemical information is neglected. As a further simplification, models were fitted using descriptors from just a few atomic positions occupied in the majority of active or inactive compounds. No further loss in performance was observed, even though in one case descriptors from a single atomic position were used, and in all cases the number of atomic positions required was fewer than twelve. The resulting models suggest that simply filling space at a few key atomic positions is responsible for enhanced activity. At least for the steroids, this finding is at odds with known SAR and binding interactions with the relevant receptor. Using a simulated data set, we illustrate that this paradoxical outcome is a symptom of having too few observations to describe the response in a data set. It is concluded that none of the nine data sets examined can reliably distinguish the merits of different 3D QSAR descriptors and that they should not be used for this purpose. We advocate the use of simulated data, instead.