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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Treatment of prolapsed gland and cartilage deformity of the nictitating membrane with pocket technique and chondrectomy alone, or combined with a wedge conjunctivectomy: 132 dogs (1998-2018).

OBJECTIVE: To evaluate the results obtained using Morgan pocket technique and chondrectomy (MPTC) alone, or combined with a wedge conjunctivectomy (MPTC + WC) for the treatment of prolapsed gland associated with cartilage deformity of the nictitating membrane (PGCD) in dogs. PROCEDURE: Medical records of dogs diagnosed with PGCD that received a MPTC or MPTC + WC were reviewed between 1998 and 2018. Success rate was defined by lack of recurrence of the prolapsed gland with a minimum of 6 months follow-up. Histological examination of the excised cartilage was performed in 13 eyes. RESULTS: A total of 132 dogs (181 eyes) met the inclusion criteria. Median follow-up time was 25 months (range, 6-166 months). MPTC was used in 131 eyes with 91.6% success rate. MPTC + WC was used in 50 eyes with 100% success rate. Postoperative complications occurred in 6.9% and 4%, respectively, for MPTC and MPTC + WC, including lacrimal cysts (8 eyes) or corneal erosion (3 eyes). Recurrence and complications rates were significantly lower using MPTC + WC compared with MPTC in the giant breed dogs (P = .019 and P = .002, respectively), but not in the overall study population (P = .328 and P = .290, respectively). Histological cartilage anomalies were noted in 2/13 specimens from chronic PGCD. CONCLUSIONS: MPTC + WC offers a good therapeutic option for giant breed dogs with PGCD. The combined technique provides a good apposition and mobility of the nictitating membrane onto the ocular surface and potentially reduces the risk of recurrence in these giant canine breeds.

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.

Blinded predictions of standard binding free energies: lessons learned from the SAMPL6 challenge.

In the context of the SAMPL6 challenges, series of blinded predictions of standard binding free energies were made with the SOMD software for a dataset of 27 host-guest systems featuring two octa-acids hosts (OA and TEMOA) and a cucurbituril ring (CB8) host. Three different models were used, ModelA computes the free energy of binding based on a double annihilation technique; ModelB additionally takes into account long-range dispersion and standard state corrections; ModelC additionally introduces an empirical correction term derived from a regression analysis of SAMPL5 predictions previously made with SOMD. The performance of each model was evaluated with two different setups; buffer explicitly matches the ionic strength from the binding assays, whereas no-buffer merely neutralizes the host-guest net charge with counter-ions. ModelC/no-buffer shows the lowest mean-unsigned error for the overall dataset (MUE 1.29 < 1.39 < 1.50 kcal mol-1, 95% CI), while explicit modelling of the buffer improves significantly results for the CB8 host only. Correlation with experimental data ranges from excellent for the host TEMOA (R2 0.91 < 0.94 < 0.96), to poor for CB8 (R2 0.04 < 0.12 < 0.23). Further investigations indicate a pronounced dependence of the binding free energies on the modelled ionic strength, and variable reproducibility of the binding free energies between different simulation packages.

Impact of domain knowledge on blinded predictions of binding energies by alchemical free energy calculations.

The Drug Design Data Resource (D3R) consortium organises blinded challenges to address the latest advances in computational methods for ligand pose prediction, affinity ranking, and free energy calculations. Within the context of the second D3R Grand Challenge several blinded binding free energies predictions were made for two congeneric series of Farsenoid X Receptor (FXR) inhibitors with a semi-automated alchemical free energy calculation workflow featuring FESetup and SOMD software tools. Reasonable performance was observed in retrospective analyses of literature datasets. Nevertheless, blinded predictions on the full D3R datasets were poor due to difficulties encountered with the ranking of compounds that vary in their net-charge. Performance increased for predictions that were restricted to subsets of compounds carrying the same net-charge. Disclosure of X-ray crystallography derived binding modes maintained or improved the correlation with experiment in a subsequent rounds of predictions. The best performing protocols on D3R set1 and set2 were comparable or superior to predictions made on the basis of analysis of literature structure activity relationships (SAR)s only, and comparable or slightly inferior, to the best submissions from other groups.

Blinded predictions of host-guest standard free energies of binding in the SAMPL5 challenge.

In the context of the SAMPL5 blinded challenge standard free energies of binding were predicted for a dataset of 22 small guest molecules and three different host molecules octa-acids (OAH and OAMe) and a cucurbituril (CBC). Three sets of predictions were submitted, each based on different variations of classical molecular dynamics alchemical free energy calculation protocols based on the double annihilation method. The first model (model A) yields a free energy of binding based on computed free energy changes in solvated and host-guest complex phases; the second (model B) adds long range dispersion corrections to the previous result; the third (model C) uses an additional standard state correction term to account for the use of distance restraints during the molecular dynamics simulations. Model C performs the best in terms of mean unsigned error for all guests (MUE [Formula: see text]-95 % confidence interval) for the whole data set and in particular for the octa-acid systems (MUE [Formula: see text]). The overall correlation with experimental data for all models is encouraging ([Formula: see text]). The correlation between experimental and computational free energy of binding ranks as one of the highest with respect to other entries in the challenge. Nonetheless the large MUE for the best performing model highlights systematic errors, and submissions from other groups fared better with respect to this metric.

Impact of Ser17 Phosphorylation on the Conformational Dynamics of the Oncoprotein MDM2.

MDM2 is an important oncoprotein that downregulates the activity of the tumor suppressor protein p53 via binding of its N-terminal domain to the p53 transactivation domain. The first 24 residues of the MDM2 N-terminal domain form an intrinsically disordered "lid" region that interconverts on a millisecond time scale between "open" and "closed" states in unliganded MDM2. While the former conformational state is expected to facilitate p53 binding, the latter competes in a pseudo-substrate manner with p53 for its binding site. Phosphorylation of serine 17 in the MDM2 lid region is thought to modulate the equilibrium between "open" and "closed" lid states, but contradictory findings on the favored lid conformational state upon phosphorylation have been reported. Here, the nature of the conformational states of MDM2 pSer17 and Ser17Asp variants was addressed by means of enhanced sampling molecular dynamics simulations. Detailed analyses of the computed lid conformational ensembles indicate that both lid variants stabilize a "closed" state, with respect to wild type. Nevertheless, the nature of the closed-state conformational ensembles differs significantly between the pSer17 and Ser17Asp variants. Thus, care should be applied in the interpretation of biochemical experiments that use phosphomimetic variants to model the effects of phosphorylation on the structure and dynamics of this disordered protein region.

Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2.

Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29) peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2.

Blinded predictions of distribution coefficients in the SAMPL5 challenge.

In the context of the SAMPL5 challenge water-cyclohexane distribution coefficients for 53 drug-like molecules were predicted. Four different models based on molecular dynamics free energy calculations were tested. All models initially assumed only one chemical state present in aqueous or organic phases. Model A is based on results from an alchemical annihilation scheme; model B adds a long range correction for the Lennard Jones potentials to model A; model C adds charging free energy corrections; model D applies the charging correction from model C to ionizable species only. Model A and B perform better in terms of mean-unsigned error ([Formula: see text] D units - 95 % confidence interval) and determination coefficient [Formula: see text], while charging corrections lead to poorer results with model D ([Formula: see text] and [Formula: see text]). Because overall errors were large, a retrospective analysis that allowed co-existence of ionisable and neutral species of a molecule in aqueous phase was investigated. This considerably reduced systematic errors ([Formula: see text] and [Formula: see text]). Overall accurate [Formula: see text] predictions for drug-like molecules that may adopt multiple tautomers and charge states proved difficult, indicating a need for methodological advances to enable satisfactory treatment by explicit-solvent molecular simulations.

Blinded predictions of binding modes and energies of HSP90-α ligands for the 2015 D3R grand challenge.

In the framework of the 2015 D3R inaugural grand challenge, blind binding pose and affinity predictions were performed for a set of 180 ligands of the Heat Shock Protein HSP90-α protein, a relevant cancer target. Spectral clustering was used to rapidly identify alternative binding site conformations in publicly available crystallographic HSP90-α structures. Subsequently, multiple docking and scoring protocols employing the software Autodock Vina and rDock were applied to predict binding modes and rank order ligands. Alchemical free energy calculations were performed with the software FESetup and Sire/OpenMM to predict binding affinities for three congeneric series subsets. Some of the protocols used here were ranked among the top submissions according to most of the evaluation metrics. Docking performance was excellent, but the scoring results were disappointing. A critical assessment of the results is reported, as well as suggestions for future similar competitions.