Sire: An interoperability engine for prototyping algorithms and exchanging information between molecular simulation programs.

Sire is a Python/C++ library that is used both to prototype new algorithms and as an interoperability engine for exchanging information between molecular simulation programs. It provides a collection of file parsers and information converters that together make it easier to combine and leverage the functionality of many other programs and libraries. This empowers researchers to use sire to write a single script that can, for example, load a molecule from a PDBx/mmCIF file via Gemmi, perform SMARTS searches via RDKit, parameterize molecules using BioSimSpace, run GPU-accelerated molecular dynamics via OpenMM, and then display the resulting dynamics trajectory in a NGLView Jupyter notebook 3D molecular viewer. This functionality is built on by BioSimSpace, which uses sire's molecular information engine to interconvert with programs such as GROMACS, NAMD, Amber, and AmberTools for automated molecular parameterization and the running of molecular dynamics, metadynamics, and alchemical free energy workflows. Sire comes complete with a powerful molecular information search engine, plus trajectory loading and editing, analysis, and energy evaluation engines. This, when combined with an in-built computer algebra system, gives substantial flexibility to researchers to load, search for, edit, and combine molecular information from multiple sources and use that to drive novel algorithms by combining functionality from other programs. Sire is open source (GPL3) and is available via conda and at a free Jupyter notebook server at https://try.openbiosim.org. Sire is supported by the not-for-profit OpenBioSim community interest company.

OpenMM 8: Molecular Dynamics Simulation with Machine Learning Potentials.

Machine learning plays an important and growing role in molecular simulation. The newest version of the OpenMM molecular dynamics toolkit introduces new features to support the use of machine learning potentials. Arbitrary PyTorch models can be added to a simulation and used to compute forces and energy. A higher-level interface allows users to easily model their molecules of interest with general purpose, pretrained potential functions. A collection of optimized CUDA kernels and custom PyTorch operations greatly improves the speed of simulations. We demonstrate these features in simulations of cyclin-dependent kinase 8 (CDK8) and the green fluorescent protein chromophore in water. Taken together, these features make it practical to use machine learning to improve the accuracy of simulations with only a modest increase in cost.

OpenMM 8: Molecular Dynamics Simulation with Machine Learning Potentials.

Machine learning plays an important and growing role in molecular simulation. The newest version of the OpenMM molecular dynamics toolkit introduces new features to support the use of machine learning potentials. Arbitrary PyTorch models can be added to a simulation and used to compute forces and energy. A higher-level interface allows users to easily model their molecules of interest with general purpose, pretrained potential functions. A collection of optimized CUDA kernels and custom PyTorch operations greatly improves the speed of simulations. We demonstrate these features on simulations of cyclin-dependent kinase 8 (CDK8) and the green fluorescent protein (GFP) chromophore in water. Taken together, these features make it practical to use machine learning to improve the accuracy of simulations at only a modest increase in cost.

Deconstructing allostery by computational assessment of the binding determinants of allosteric PTP1B modulators.

Fragment-based drug discovery is an established methodology for finding hit molecules that can be elaborated into lead compounds. However it is currently challenging to predict whether fragment hits that do not bind to an orthosteric site could be elaborated into allosteric modulators, as in these cases binding does not necessarily translate into a functional effect. We propose a workflow using Markov State Models (MSMs) with steered molecular dynamics (sMD) to assess the allosteric potential of known binders. sMD simulations are employed to sample protein conformational space inaccessible to routine equilibrium MD timescales. Protein conformations sampled by sMD provide starting points for seeded MD simulations, which are combined into MSMs. The methodology is demonstrated on a dataset of protein tyrosine phosphatase 1B ligands. Experimentally confirmed allosteric inhibitors are correctly classified as inhibitors, whereas the deconstructed analogues show reduced inhibitory activity. Analysis of the MSMs provide insights into preferred protein-ligand arrangements that correlate with functional outcomes. The present methodology may find applications for progressing fragments towards lead molecules in FBDD campaigns.

Comparison of Receptor-Ligand Restraint Schemes for Alchemical Absolute Binding Free Energy Calculations.

Alchemical absolute binding free energy calculations are of increasing interest in drug discovery. These calculations require restraints between the receptor and ligand to restrict their relative positions and, optionally, orientations. Boresch restraints are commonly used, but they must be carefully selected in order to sufficiently restrain the ligand and to avoid inherent instabilities. Applying multiple distance restraints between anchor points in the receptor and ligand provides an alternative framework without inherent instabilities which may provide convergence benefits by more strongly restricting the relative movements of the receptor and ligand. However, there is no simple method to calculate the free energy of releasing these restraints due to the coupling of the internal and external degrees of freedom of the receptor and ligand. Here, a method to rigorously calculate free energies of binding with multiple distance restraints by imposing intramolecular restraints on the anchor points is proposed. Absolute binding free energies for the human macrophage migration inhibitory factor/MIF180, system obtained using a variety of Boresch restraints and rigorous and nonrigorous implementations of multiple distance restraints are compared. It is shown that several multiple distance restraint schemes produce estimates in good agreement with Boresch restraints. In contrast, calculations without orientational restraints produce erroneously favorable free energies of binding by up to approximately 4 kcal mol-1. These approaches offer new options for the deployment of alchemical absolute binding free energy calculations.

Energetics of a protein disorder-order transition in small molecule recognition.

Many proteins recognise other proteins via mechanisms that involve the folding of intrinsically disordered regions upon complex formation. Here we investigate how the selectivity of a drug-like small molecule arises from its modulation of a protein disorder-to-order transition. Binding of the compound AM-7209 has been reported to confer order upon an intrinsically disordered 'lid' region of the oncoprotein MDM2. Calorimetric measurements revealed that truncation of the lid region of MDM2 increases the apparent dissociation constant of AM-7209 250-fold. By contrast, lid truncation has little effect on the binding of the ligand Nutlin-3a. Insights into these differential binding energetics were obtained via a complete thermodynamic analysis that featured adaptive absolute alchemical free energy of binding calculations with enhanced-sampling molecular dynamics simulations. The simulations reveal that in apo MDM2 the ordered lid state is energetically disfavoured. AM-7209, but not Nutlin-3a, shows a significant energetic preference for ordered lid conformations, thus shifting the balance towards ordering of the lid in the AM-7209/MDM2 complex. The methodology reported herein should facilitate broader targeting of intrinsically disordered regions in medicinal chemistry.

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies.

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.

Ocular dermoids in 13 cats: a multicentre retrospective study.

OBJECTIVES: The aim of this multicentre retrospective study was to review the clinical data, outcomes and histopathological features of cats that had been treated for ocular surface dermoids. METHODS: Thirteen cats from various private practices in France with a clinical diagnosis of ocular surface dermoid were included in the study. RESULTS: The mean age of the study population at the time of diagnosis was 5 months. There were nine males and four females. Three different breeds were domestic shorthair (n = 7), Birman (n = 4) and Havana Brown (n = 2). Two of the four Birmans were related (same sire). The two Havana Browns were also related (same sire). All of the dermoids were unilateral. Five of the dermoids were strictly conjunctival. Four affected both the conjunctiva and the cornea. Three affected both the conjunctiva and the eyelid, and one was strictly corneal. They were located in various positions: temporal (n = 9), inferonasal (n = 1), dorsonasal (n = 1) and dorsotemporal (n = 1). The last dermoid was heterogeneous and involved the nasal, dorsal and temporal quadrants. Concurrent eye diseases were observed in five patients: four cats exhibited associated eyelid agenesis and one cat exhibited persistent iris-to-iris pupillary membranes. Ten dermoids were surgically excised with no recurrences. Surgery was not performed for three cats: one cat died a few days after diagnosis and two cats were lost to follow-up after initial presentation. CONCLUSIONS AND RELEVANCE: Ocular surface dermoids are a rare condition in cats that can be treated successfully by surgical excision. Although our study reports only a small number of cases, the observation of ocular surface dermoids in two related cats in two different breeds indicates that genetic transmission is likely.

Ectopic cilia in 112 dogs: A multicenter retrospective study.

OBJECTIVE: The aim of this retrospective study was to review the clinical data and outcomes of patients that suffered ectopic cilium (EC). ANIMALS STUDIED: One hundred and twelve dogs from multiple private practices in France, with a clinical diagnosis of EC were included in the study. RESULTS: The mean age of affected dogs was 2.3 years. There were 64 females and 48 males. The most represented breeds were the Shi Tzu, the French Bulldog, the English Bulldog and the Chihuahua. Eleven dogs were affected bilaterally. The upper eyelid was implicated in 93.5% of the cases, with the median portion being the most affected. No statistical difference was observed between the right and the left eye. EC were associated with distichiasis in 50% of the cases. Pigmentation of the conjunctiva at the point of exit of the EC was present in 58% of the cases. EC were short in 75% and long in 25% of the cases. Corneal complications were statistically associated with short EC. The corneal lesions associated with EC were keratitis (94%), corneal granuloma (0.8%), corneal fibrosis (2.7%), corneal degeneration (0.8%), superficial corneal ulcer (68.7%), deep corneal ulcer (8%) and perforating corneal ulcer (0.8%). The surgeries which consisted of the removal of the hair follicle was successful in 88.4% of the cases. CONCLUSION: EC is a rare condition which can be treated successfully by the removal of the hair follicles. It must be suspected in cases of corneal lesions unresponsive to medical treatment.

IL-15 Prevents Renal Fibrosis by Inhibiting Collagen Synthesis: A New Pathway in Chronic Kidney Disease?

Chronic kidney disease (CKD), secondary to renal fibrogenesis, is a public health burden. The activation of interstitial myofibroblasts and excessive production of extracellular matrix (ECM) proteins are major events leading to end-stage kidney disease. Recently, interleukin-15 (IL-15) has been implicated in fibrosis protection in several organs, with little evidence in the kidney. Since endogenous IL-15 expression decreased in nephrectomized human allografts evolving toward fibrosis and kidneys in the unilateral ureteral obstruction (UUO) model, we explored IL-15's renoprotective role by pharmologically delivering IL-15 coupled or not with its soluble receptor IL-15Rα. Despite the lack of effects on myofibroblast accumulation, both IL-15 treatments prevented tubulointerstitial fibrosis (TIF) in UUO as characterized by reduced collagen and fibronectin deposition. Moreover, IL-15 treatments inhibited collagen and fibronectin secretion by transforming growth factor-ß (TGF-ß)-treated primary myofibroblast cultures, demonstrating that the antifibrotic effect of IL-15 in UUO acts, in part, through a direct inhibition of ECM synthesis by myofibroblasts. In addition, IL-15 treatments resulted in decreased expression of monocyte chemoattractant protein 1 (MCP-1) and subsequent macrophage infiltration in UUO. Taken together, our study highlights a major role of IL-15 on myofibroblasts and macrophages, two main effector cells in renal fibrosis, demonstrating that IL-15 may represent a new therapeutic option for CKD.

Autologous lamellar keratoplasty for the treatment of feline corneal sequestrum: A retrospective study of 35 eyes (2012-2020).

OBJECTIVE: To evaluate the use of autologous lamellar keratoplasty for the treatment of feline corneal sequestrum (FCS). PROCEDURE: The medical records of cats diagnosed with FCS that underwent autologous lamellar keratoplasty between 2012 and 2020 with a minimum of 2 months of follow-up were reviewed. After keratectomy of FCS, a button adjacent to the corneal limbus was harvested on the same eye and sutured to the recipient bed. A nictitating membrane flap was left in place until the first recheck except for one patient. Postoperative treatment with topical and systemic antibiotics and systemic nonsteroidal anti-inflammatory medications was prescribed. Follow-up examinations were carried out 2 weeks, 1 month and 2 months post-operatively and consisted of a complete ophthalmic examination. RESULTS: A total of 35 cats (35 eyes) were included. The median follow-up time was 3.2 months (range, 2-59 months). Brachycephalic cats were overrepresented (85.7%). The mean graft size was 6.5 mm (range, 6-9 mm). Minor complications consisting of melting and partial integration of the graft occurred in 2/35 eyes (5.7%). Recurrence was observed in 1/35 eyes (2.9%) and was managed by a superficial keratectomy. A good visual outcome was achieved in all eyes, and a faint or mild corneal opacification occurred in 15/35 (42.9%). CONCLUSIONS: Autologous lamellar keratoplasty is an effective treatment for FCS, providing good tectonic support to the affected cornea and resulting in good visual and cosmetic outcomes. These results should be verified in future prospective studies that include a larger number of cases and longer-term follow-up.

BMAL1 Knockdown Leans Epithelial-Mesenchymal Balance toward Epithelial Properties and Decreases the Chemoresistance of Colon Carcinoma Cells.

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial-mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial-mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.

Implementation of the QUBE Force Field in SOMD for High-Throughput Alchemical Free-Energy Calculations.

The quantum mechanical bespoke (QUBE) force-field approach has been developed to facilitate the automated derivation of potential energy function parameters for modeling protein-ligand binding. To date, the approach has been validated in the context of Monte Carlo simulations of protein-ligand complexes. We describe here the implementation of the QUBE force field in the alchemical free-energy calculation molecular dynamics simulation package SOMD. The implementation is validated by demonstrating the reproducibility of absolute hydration free energies computed with the QUBE force field across the SOMD and GROMACS software packages. We further demonstrate, by way of a case study involving two series of non-nucleoside inhibitors of HIV-1 reverse transcriptase, that the availability of QUBE in a modern simulation package that makes efficient use of graphics processing unit acceleration will facilitate high-throughput alchemical free-energy calculations.

Combining Virtual Reality Visualization with Ensemble Molecular Dynamics to Study Complex Protein Conformational Changes.

Molecular dynamics (MD) simulations are increasingly used to elucidate relationships between protein structure, dynamics, and their biological function. Currently, it is extremely challenging to perform MD simulations of large-scale structural rearrangements in proteins that occur on millisecond timescales or beyond, as this requires very significant computational resources, or the use of cumbersome "collective variable" enhanced sampling protocols. Here, we describe a framework that combines ensemble MD simulations and virtual reality visualization (eMD-VR) to enable users to interactively generate realistic descriptions of large amplitude, millisecond timescale protein conformational changes in proteins. Detailed tests demonstrate that eMD-VR substantially decreases the computational cost of folding simulations of a WW domain, without the need to define collective variables a priori. We further show that eMD-VR generated pathways can be combined with Markov state models to describe the thermodynamics and kinetics of large-scale loop motions in the enzyme cyclophilin A. Our results suggest eMD-VR is a powerful tool for exploring protein energy landscapes in bioengineering efforts.

Corneal epithelial inclusion cysts in 12 dogs (13 eyes) from 2010 to 2019: A multicentric retrospective study.

OBJECTIVE: The aim of this retrospective study was to review the clinical data and outcomes of patients that suffered corneal epithelial inclusion cysts (CEIC). Animals studied Thirteen eyes from 12 dogs from multiple private practices in France and Belgium, with a strong clinical of CEIC were included in the study. RESULTS: The mean age of affected dogs was 9 years. There were 9 females and 3 males. Two out of 12 dogs were Shih Tzus. Where identified, etiology was traumatic. Eleven dogs were affected unilaterally, 1 bilaterally. The cysts were single, bi-lobulated, or tri-lobulate, and cyst size ranged from 1 to 6 mm in diameter. Keratectomy alone was performed in seven cases, in conjunction with a conjunctival graft in three cases, a porcine small intestine mucosa graft in two cases, or an amniotic membrane graft in one case. Histopathologic findings were consistent in all eyes with a cyst located in the corneal stroma, composed of a nonkeratinized squamous epithelium delineating a lumen filled with neutrophils and desquamated epithelial cells. Surgery was curative in all cases but one, where the removal was incomplete and recurrence occurred several weeks after the surgery. CONCLUSION: Corneal epithelial inclusion cysts is a rare condition, secondary to corneal trauma, which can be treated successfully with conventional keratectomy alone or in conjunction with graft procedures.

Hybrid Alchemical Free Energy/Machine-Learning Methodology for the Computation of Hydration Free Energies.

A methodology that combines alchemical free energy calculations (FEP) with machine learning (ML) has been developed to compute accurate absolute hydration free energies. The hybrid FEP/ML methodology was trained on a subset of the FreeSolv database and retrospectively shown to outperform most submissions from the SAMPL4 competition. Compared to pure machine-learning approaches, FEP/ML yields more precise estimates of free energies of hydration and requires a fraction of the training set size to outperform standalone FEP calculations. The ML-derived correction terms are further shown to be transferable to a range of related FEP simulation protocols. The approach may be used to inexpensively improve the accuracy of FEP calculations and to flag molecules which will benefit the most from bespoke force field parametrization efforts.

Assessment of Binding Affinity via Alchemical Free-Energy Calculations.

Free-energy calculations have seen increased usage in structure-based drug design. Despite the rising interest, automation of the complex calculations and subsequent analysis of their results are still hampered by the restricted choice of available tools. In this work, an application for automated setup and processing of free-energy calculations is presented. Several sanity checks for assessing the reliability of the calculations were implemented, constituting a distinct advantage over existing open-source tools. The underlying workflow is built on top of the software Sire, SOMD, BioSimSpace, and OpenMM and uses the AMBER 14SB and GAFF2.1 force fields. It was validated on two datasets originally composed by Schrödinger, consisting of 14 protein structures and 220 ligands. Predicted binding affinities were in good agreement with experimental values. For the larger dataset, the average correlation coefficient Rp was 0.70 ± 0.05 and average Kendall's τ was 0.53 ± 0.05, which are broadly comparable to or better than previously reported results using other methods.

BMAL1 knockdown triggers different colon carcinoma cell fates by altering the delicate equilibrium between AKT/mTOR and P53/P21 pathways.

Dysregulation of the circadian timing system (CTS) frequently appears during colorectal cancer (CRC) progression. In order to better understand the role of the circadian clock in CRC progression, this study evaluated in vitro how knockdown of a core circadian protein BMAL1 (BMAL1-KD) influenced the behavior of two primary human CRC cell lines (HCT116 and SW480) and a metastatic CRC cell line (SW620).Unexpectedly, BMAL1-KD induced CRC cell-type specific responses rather than the same phenomenon throughout. First, BMAL1-KD increased AKT/mTOR activation in each CRC cell line, but to different extents. Second, BMAL1-KD-induced P53 activation varied with cell context. In a wild type P53 background, HCT116 BMAL1-KD cells quickly underwent apoptosis after shBMAL1 lentivirus transduction, while surviving cells showed less P53 but increased AKT/mTOR activation, which ultimately caused higher proliferation. In the presence of a partially functional mutant P53, SW480 BMAL1-KD cells showed moderate P53 and mTOR activation simultaneously with cell senescence. With a moderate increased AKT but unchanged mutant P53 activation, SW620 BMAL1-KD cells grew faster.Thus, under different CRC cellular pathological contexts, BMAL1 knockdown induced relatively equal effects on AKT/mTOR activation but different effects on P53 activation, which finally triggered different CRC cell fates.

Investigating Cryptic Binding Sites by Molecular Dynamics Simulations.

This Account highlights recent advances and discusses major challenges in investigations of cryptic (hidden) binding sites by molecular simulations. Cryptic binding sites are not visible in protein targets crystallized without a ligand and only become visible crystallographically upon binding events. These sites have been shown to be druggable and might provide a rare opportunity to target difficult proteins. However, due to their hidden nature, they are difficult to find through experimental screening. Computational methods based on atomistic molecular simulations remain one of the best approaches to identify and characterize cryptic binding sites. However, not all methods are equally efficient. Some are more apt at quickly probing protein dynamics but do not provide thermodynamic or druggability information, while others that are able to provide such data are demanding in terms of time and resources. Here, we review the recent contributions of mixed-solvent simulations, metadynamics, Markov state models, and other enhanced sampling methods to the field of cryptic site identification and characterization. We discuss how these methods were able to provide precious information on the nature of the site opening mechanisms, to predict previously unknown sites which were used to design new ligands, and to compute the free energy landscapes and kinetics associated with the opening of the sites and the binding of the ligands. We highlight the potential and the importance of such predictions in drug discovery, especially for difficult ("undruggable") targets. We also discuss the major challenges in the field and their possible solutions.

The SAMPL6 SAMPLing challenge: assessing the reliability and efficiency of binding free energy calculations.

Approaches for computing small molecule binding free energies based on molecular simulations are now regularly being employed by academic and industry practitioners to study receptor-ligand systems and prioritize the synthesis of small molecules for ligand design. Given the variety of methods and implementations available, it is natural to ask how the convergence rates and final predictions of these methods compare. In this study, we describe the concept and results for the SAMPL6 SAMPLing challenge, the first challenge from the SAMPL series focusing on the assessment of convergence properties and reproducibility of binding free energy methodologies. We provided parameter files, partial charges, and multiple initial geometries for two octa-acid (OA) and one cucurbit[8]uril (CB8) host-guest systems. Participants submitted binding free energy predictions as a function of the number of force and energy evaluations for seven different alchemical and physical-pathway (i.e., potential of mean force and weighted ensemble of trajectories) methodologies implemented with the GROMACS, AMBER, NAMD, or OpenMM simulation engines. To rank the methods, we developed an efficiency statistic based on bias and variance of the free energy estimates. For the two small OA binders, the free energy estimates computed with alchemical and potential of mean force approaches show relatively similar variance and bias as a function of the number of energy/force evaluations, with the attach-pull-release (APR), GROMACS expanded ensemble, and NAMD double decoupling submissions obtaining the greatest efficiency. The differences between the methods increase when analyzing the CB8-quinine system, where both the guest size and correlation times for system dynamics are greater. For this system, nonequilibrium switching (GROMACS/NS-DS/SB) obtained the overall highest efficiency. Surprisingly, the results suggest that specifying force field parameters and partial charges is insufficient to generally ensure reproducibility, and we observe differences between seemingly converged predictions ranging approximately from 0.3 to 1.0 kcal/mol, even with almost identical simulations parameters and system setup (e.g., Lennard-Jones cutoff, ionic composition). Further work will be required to completely identify the exact source of these discrepancies. Among the conclusions emerging from the data, we found that Hamiltonian replica exchange-while displaying very small variance-can be affected by a slowly-decaying bias that depends on the initial population of the replicas, that bidirectional estimators are significantly more efficient than unidirectional estimators for nonequilibrium free energy calculations for systems considered, and that the Berendsen barostat introduces non-negligible artifacts in expanded ensemble simulations.