Correct Nucleotide Selection Is Confined at the Binding Site of Polymerase Enzymes.

DNA polymerases (Pols) add incoming nucleotides (deoxyribonucleoside triphosphate (dNTPs)) to growing DNA strands, a crucial step for DNA synthesis. The insertion of correct (vs incorrect) nucleotides relates to Pols' fidelity, which defines Pols' ability to faithfully replicate DNA strands in a template-dependent manner. We and others have demonstrated that reactant alignment and correct base pairing at the Pols catalytic site are crucial structural features to fidelity. Here, we first used equilibrium molecular simulations to demonstrate that the local dynamics at the protein-DNA interface in the proximity of the catalytic site is different when correct vs incorrect dNTPs are bound to polymerase ß (Pol ß). Formation and dynamic stability of specific interatomic interactions around the incoming nucleotide influence the overall binding site architecture. This explains why certain Pols' mutants can affect the local catalytic environment and influence the selection of correct vs incorrect nucleotides. In particular, this is here demonstrated by analyzing the interaction network formed by the residue R283, whose mutant R283A has an experimentally measured lower capacity of differentiating correct (G:dCTP) vs incorrect (G:dATP) base pairing in Pol ß. We also used alchemical free-energy calculations to quantify the G:dCTP →G:dATP transformation in Pol ß wild-type and mutant R283A. These results correlate well with the experimental trend, thus corroborating our mechanistic insights. Sequence and structural comparisons with other Pols from the same family suggest that these findings may also be valid in similar enzymes.

Discovery of CDC42 Inhibitors with a Favorable Pharmacokinetic Profile and Anticancer In Vivo Efficacy.

We previously reported trisubstituted pyrimidine lead compounds, namely, ARN22089 and ARN25062, which block the interaction between CDC42 with its specific downstream effector, a PAK protein. This interaction is crucial for the progression of multiple tumor types. Such inhibitors showed anticancer efficacy in vivo. Here, we describe a second class of CDC42 inhibitors with favorable drug-like properties. Out of the 25 compounds here reported, compound 15 (ARN25499) stands out as the best lead compound with an improved pharmacokinetic profile, increased bioavailability, and efficacy in an in vivo PDX tumor mouse model. Our results indicate that these CDC42 inhibitors represent a promising chemical class toward the discovery of anticancer drugs, with ARN25499 as an additional lead candidate for preclinical development.

Targeting the conserved active site of splicing machines with specific and selective small molecule modulators.

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.

Tumor growth-arrest effect of tetrahydroquinazoline-derivative human topoisomerase II-alpha inhibitor in HPV-negative head and neck squamous cell carcinoma.

Oral malignancies continue to have severe morbidity with less than 50% long-term survival despite the advancement in the available therapies. There is a persisting demand for new approaches to establish more efficient strategies for their treatment. In this regard, the human topoisomerase II (topoII) enzyme is a validated chemotherapeutics target, as topoII regulates vital cellular processes such as DNA replication, transcription, recombination, and chromosome segregation in cells. TopoII inhibitors are currently used to treat some neoplasms such as breast and small cells lung carcinomas. Additionally, topoII inhibitors are under investigation for the treatment of other cancer types, including oral cancer. Here, we report the therapeutic effect of a tetrahydroquinazoline derivative (named ARN21934) that preferentially inhibits the alpha isoform of human topoII. The treatment efficacy of ARN21934 has been evaluated in 2D cell cultures, 3D in vitro systems, and in chick chorioallantoic membrane cancer models. Overall, this work paves the way for further preclinical developments of ARN21934 and possibly other topoII alpha inhibitors of this promising chemical class as a new chemotherapeutic approach for the treatment of oral neoplasms.

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism.

The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.

Molecular Mechanisms Underlying Detection Sensitivity in Nanoparticle-Assisted NMR Chemosensing.

Nanoparticle-assisted nuclear magnetic resonance (NMR) chemosensing exploits monolayer-protected nanoparticles as supramolecular hosts to detect small molecules in complex mixtures via nuclear Overhauser effect experiments with detection limits down to the micromolar range. Still, the structure-sensitivity relationships at the basis of such detection limits are little understood. In this work, we integrate NMR spectroscopy and atomistic molecular dynamics simulations to examine the covariates that affect the sensitivity of different NMR chemosensing experiments [saturation transfer difference (STD), water STD, and high-power water-mediated STD]. Our results show that the intensity of the observed signals correlates with the number and duration of the spin-spin interactions between the analytes and the nanoparticles and/or between the analytes and the nanoparticles' solvation molecules. In turn, these parameters depend on the location and dynamics of each analyte inside the monolayer. This insight will eventually facilitate the tailoring of experimental and computational setups to the analyte's chemistry, making NMR chemosensing an even more effective technique in practical use.

Combining structural and coevolution information to unveil allosteric sites.

Understanding allosteric regulation in biomolecules is of great interest to pharmaceutical research and computational methods emerged during the last decades to characterize allosteric coupling. However, the prediction of allosteric sites in a protein structure remains a challenging task. Here, we integrate local binding site information, coevolutionary information, and information on dynamic allostery into a structure-based three-parameter model to identify potentially hidden allosteric sites in ensembles of protein structures with orthosteric ligands. When tested on five allosteric proteins (LFA-1, p38-α, GR, MAT2A, and BCKDK), the model successfully ranked all known allosteric pockets in the top three positions. Finally, we identified a novel druggable site in MAT2A confirmed by X-ray crystallography and SPR and a hitherto unknown druggable allosteric site in BCKDK validated by biochemical and X-ray crystallography analyses. Our model can be applied in drug discovery to identify allosteric pockets.

Drug Design in the Exascale Era: A Perspective from Massively Parallel QM/MM Simulations.

The initial phases of drug discovery - in silico drug design - could benefit from first principle Quantum Mechanics/Molecular Mechanics (QM/MM) molecular dynamics (MD) simulations in explicit solvent, yet many applications are currently limited by the short time scales that this approach can cover. Developing scalable first principle QM/MM MD interfaces fully exploiting current exascale machines - so far an unmet and crucial goal - will help overcome this problem, opening the way to the study of the thermodynamics and kinetics of ligand binding to protein with first principle accuracy. Here, taking two relevant case studies involving the interactions of ligands with rather large enzymes, we showcase the use of our recently developed massively scalable Multiscale Modeling in Computational Chemistry (MiMiC) QM/MM framework (currently using DFT to describe the QM region) to investigate reactions and ligand binding in enzymes of pharmacological relevance. We also demonstrate for the first time strong scaling of MiMiC-QM/MM MD simulations with parallel efficiency of ∼70% up to >80,000 cores. Thus, among many others, the MiMiC interface represents a promising candidate toward exascale applications by combining machine learning with statistical mechanics based algorithms tailored for exascale supercomputers.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

On the Role of Molecular Conformation of the 8-Oxoguanine Lesion in Damaged DNA Processing by Polymerases.

A common and insidious DNA damage is 8-oxoguanine (8OG), bypassed with low catalytic efficiency and high error frequency by polymerases (Pols) during DNA replication. This is a fundamental process with far-reaching implications in cell function and diseases. However, the molecular determinants of how 8OG exactly affects the catalytic efficiency of Pols remain largely unclear. By examining ternary deoxycytidine triphosphate/DNA/Pol complexes containing the 8OG damage, we found that 8OG consistently adopts different conformations when bound to Pols, compared to when in isolated DNA. Equilibrium molecular dynamics and metadynamics free energy calculations quantified that 8OG is in the lowest energy conformation in isolated DNA. In contrast, 8OG adopts high-energy conformations often characterized by intramolecular steric repulsion when bound to Pols. We show that the 8OG conformation can be regulated by mutating Pol residues interacting with the 8OG phosphate group. These findings propose the 8OG conformation as a factor in Pol-mediated processing of damaged DNA.

NanoModeler CG: A Tool for Modeling and Engineering Functional Nanoparticles at a Coarse-Grained Resolution.

Functionalized metal nanoparticles (NPs) are macromolecular assemblies with a tunable physicochemical profile that makes them interesting for biotechnology, materials science, and energy conversion. In this regard, molecular simulations offer a way to scrutinize the structural and dynamical features of monolayer-protected NPs and their interactions with relevant matrices. Previously, we developed NanoModeler, a webserver that automates the preparation of functionalized gold NPs for atomistic molecular dynamics (MD) simulations. Here, we present NanoModeler CG (www.nanomodeler.it), a new release of NanoModeler that now also allows the building and parametrizing of monolayer-protected metal NPs at a coarse-grained (CG) resolution. This new version extends our original methodology to NPs of eight different core shapes, conformed by up to 800,000 beads and coated by eight different monolayer morphologies. The resulting topologies are compatible with the Martini force field but are easily extendable to any other set of parameters parsed by the user. Finally, we demonstrate NanoModeler CG's capabilities by reproducing experimental structural features of alkylthiolated NPs and rationalizing the brush-to-mushroom phase transition of PEGylated anionic NPs. By automating the construction and parametrization of functionalized NPs, the NanoModeler series offers a standardized way to computationally model monolayer-protected nanosized systems.

Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106.

The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.

Computer-aided identification, synthesis, and biological evaluation of DNA polymerase η inhibitors for the treatment of cancer.

In cancer cells, Pol η allows DNA replication and cell proliferation even in the presence of chemotherapeutic drug-induced damages, like in the case of platinum-containing drugs. Inhibition of Pol η thus represents a promising strategy to overcome drug resistance and preserve the effectiveness of chemotherapeutic drugs. Here, we report the discovery of a novel class of Pol ƞ inhibitors, with 35 active close analogs. Compound 21 (ARN24964) stands out as the best inhibitor, with an IC50 value of 14.7 µM against Pol η and a good antiproliferative activity when used in combination with cisplatin - with a synergistic effect in three different cancer cell lines (A375, A549, OVCAR3). Moreover, it is characterized by a favorable drug-like profile in terms of its aqueous kinetic solubility, plasma and metabolic stability. Thus, ARN24964 is a promising compound for further structure-based drug design efforts toward developing drugs to solve or limit the issue of drug resistance to platinum-containing drugs in cancer patients.

Design, synthesis, docking, and biochemical characterization of non-nucleoside SARS-CoV-2 RdRp inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic. The identification of effective antiviral drugs remains an urgent medical need. In this context, here we report 17 new 1,4-benzopyrone derivatives, which have been designed, synthesized, and characterized for their ability to block the RNA-dependent RNA polymerase (RdRp) enzyme, a promising target for antiviral drug discovery. This compound series represents a good starting point for developing non-nucleoside inhibitors of RdRp. Compounds 4, 5, and 8 were the most promising drug-like candidates with good potency in inhibiting RdRp, improved in vitro pharmacokinetics compared to the initial hits, and no cytotoxicity effects on normal cell (HEK-293). Compound 8 (ARN25592) stands out as the most promising inhibitor. Our results indicate that this new chemical class of 1,4-benzopyrone derivatives deserves further exploration towards novel and potent antiviral drugs for the treatment of SARS-CoV-2 and potentially other viruses.

Preclinical Development of the Na-K-2Cl Co-transporter-1 (NKCC1) Inhibitor ARN23746 for the Treatment of Neurodevelopmental Disorders.

Alterations in the expression of the Cl- importer Na-K-2Cl co-transporter-1 (NKCC1) and the exporter K-Cl co-transporter 2 (KCC2) lead to impaired intracellular chloride concentration in neurons and imbalanced excitation/inhibition in the brain. These alterations have been observed in several neurological disorders (e.g., Down syndrome and autism). Recently, we have reported the discovery of the selective NKCC1 inhibitor "compound ARN23746" for the treatment of Down syndrome and autism in mouse models. Here, we report on an extensive preclinical characterization of ARN23746 toward its development as a clinical candidate. ARN23746 shows an overall excellent metabolism profile and good brain penetration. Moreover, ARN23746 is effective in rescuing cognitive impairment in Down syndrome mice upon per os administration, in line with oral treatment of neurodevelopmental disorders. Notably, ARN23746 does not present signs of toxicity or diuresis even if administered up to 50 times the effective dose. These results further support ARN23746 as a solid candidate for clinical trial-enabling studies.

Alchemical Free-Energy Calculations of Watson-Crick and Hoogsteen Base Pairing Interconversion in DNA.

Hoogsteen (HG) base pairs have a transient nature and can be structurally similar to Watson-Crick (WC) base pairs, making their occurrence and thermodynamic stability difficult to determine experimentally. Herein, we employed the restrain-free-energy perturbation-release (R-FEP-R) method to calculate the relative free energy of the WC and HG base pairing modes in isolated and bound DNA systems and predict the glycosyl torsion conformational preference of purine bases. Notably, this method does not require prior knowledge of the transition pathway between the two end states. Remarkably, relatively fast convergence was reached, with results in excellent agreement with experimental data for all the examined DNA systems. The R-REP-R method successfully determined the stability of HG base pairing and more generally, the conformational preference of purine bases, in these systems. Therefore, this computational approach can help to understand the dynamic equilibrium between the WC and HG base pairing modes in DNA.

Alchemical Free Energy Calculations to Investigate Protein-Protein Interactions: the Case of the CDC42/PAK1 Complex.

Here, we show that alchemical free energy calculations can quantitatively compute the effect of mutations at the protein-protein interface. As a test case, we have used the protein complex formed by the small Rho-GTPase CDC42 and its downstream effector PAK1, a serine/threonine kinase. Notably, the CDC42/PAK1 complex offers a wealth of structural, mutagenesis, and binding affinity data because of its central role in cellular signaling and cancer progression. In this context, we have considered 16 mutations in the CDC42/PAK1 complex and obtained excellent agreement between computed and experimental data on binding affinity. Importantly, we also show that a careful analysis of the side-chain conformations in the mutated amino acids can considerably improve the computed estimates, solving issues related to sampling limitations. Overall, this study demonstrates that alchemical free energy calculations can conveniently be integrated into the design of experimental mutagenesis studies.

Quercetin and luteolin are single-digit micromolar inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly become a global health pandemic. Among the viral proteins, RNA-dependent RNA polymerase (RdRp) is responsible for viral genome replication and has emerged as one of the most promising targets for pharmacological intervention against SARS-CoV-2. To this end, we experimentally tested luteolin and quercetin for their ability to inhibit the RdRp enzyme. These two compounds are ancestors of flavonoid natural compounds known for a variety of basal pharmacological activities. Luteolin and quercetin returned a single-digit IC50 of 4.6 µM and 6.9 µM, respectively. Then, through dynamic docking simulations, we identified possible binding modes of these compounds to a recently published cryo-EM structure of RdRp. Collectively, these data indicate that these two compounds are a valid starting point for further optimization and development of a new class of RdRp inhibitors to treat SARS-CoV-2 and potentially other viral infections.