Targeting 7KCh-Induced Cell Death Response Mediated by p38, P2X7 and GSDME in Retinal Pigment Epithelium Cells with Sterculic Acid.

Age-related macular degeneration (AMD) is the main cause of blindness in developed countries. AMD is characterized by the formation of drusen, which are lipidic deposits, between retinal pigment epithelium (RPE) and the choroid. One of the main molecules accumulated in drusen is 7-Ketocholesterol (7KCh), an oxidized-cholesterol derivative. It is known that 7KCh induces inflammatory and cytotoxic responses in different cell types and the study of its mechanism of action is interesting in order to understand the development of AMD. Sterculic acid (SA) counteracts 7KCh response in RPE cells and could represent an alternative to improve currently used AMD treatments, which are not efficient enough. In the present study, we determine that 7KCh induces a complex cell death signaling characterized by the activation of necrosis and an alternative pyroptosis mediated by P2X7, p38 and GSDME, a new mechanism not yet related to the response to 7KCh until now. On the other hand, SA treatment can successfully attenuate the activation of both necrosis and pyroptosis, highlighting its therapeutic potential for the treatment of AMD.

pyPept: a python library to generate atomistic 2D and 3D representations of peptides.

We present pyPept, a set of executables and underlying python-language classes to easily create, manipulate, and analyze peptide molecules using the FASTA, HELM, or recently-developed BILN notations. The framework enables the analysis of both pure proteinogenic peptides as well as those with non-natural amino acids, including support to assemble a customizable monomer library, without requiring programming. From line notations, a peptide is transformed into a molecular graph for 2D depiction tasks, the calculation of physicochemical properties, and other systematic analyses or processing pipelines. The package includes a module to rapidly generate approximate peptide conformers by incorporating secondary structure restraints either given by the user or predicted via pyPept, and a wrapper tool is also provided to automate the generation and output of 2D and 3D representations of a peptide directly from the line notation. HELM and BILN notations that include circular, branched, or stapled peptides are fully supported, eliminating errors in structure creation that are prone during manual drawing and connecting. The framework and common workflows followed in pyPept are described together with illustrative examples. pyPept has been released at: https://github.com/Boehringer-Ingelheim/pyPept .

Assessing the fast prediction of peptide conformers and the impact of non-natural modifications.

We present an assessment of different approaches to predict peptide structures using modeling tools. Several small molecule, protein, and peptide-focused methodologies were used for the fast prediction of conformers for peptides shorter than 30 amino acids. We assessed the effect of including restraints based on annotated or predicted secondary structure motifs. A number of peptides in bound conformations and in solution were collected to compare the tools. In addition, we studied the impact of changing single amino acids to non-natural residues using molecular dynamics simulations. Deep learning methods such as AlphaFold2, or the combination of physics-based approaches with secondary structure information, produce the most accurate results for natural sequences. In the case of peptides with non-natural modifications, modeling the peptide containing natural amino acids first and then modifying and simulating the peptide using benchmarked force fields is a recommended pipeline. The results can guide the modeling of oligopeptides for drug discovery projects.

Toxic Determination of Cry11 Mutated Proteins Obtained Using Rational Design and Its Computational Analysis.

Cry11 proteins are toxic to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. Cry11Aa and Cry11Bb are protoxins, which when activated present their active-toxin form in two fragments between 30 and 35 kDa respectively. Previous studies conducted with Cry11Aa and Cry11Bb genes using DNA shuffling generated variant 8, which presented a deletion in the first 73 amino acids and one at position 572 and 9 substitutions including L553F and L556W. In this study, variant 8 mutants were constructed using site-directed mutagenesis, resulting in conversion of phenylalanine (F) and tryptophan (W) to leucine (L) at positions 553 and 556, respectively, producing the mutants 8F553L, 8W556L, and 8F553L/8W556L. Additionally, two mutants, A92D and C157R, derived from Cry11Bb were also generated. The proteins were expressed in the non-crystal strain BMB171 of Bacillus thuringiensis and subjected to median-lethal concentration (LC50) tests on first-instar larvae of A. aegypti. LC50 analysis showed that the 8F553L, 8W556L, 8F553L/8W556L, and C157R variants lost their toxic activity (>500 ng·mL-1), whereas the A92D protein presented a loss of toxicity of 11.4 times that of Cry11Bb. Cytotoxicity assays performed using variant 8, 8W556L and the controls Cry11Aa, Cry11Bb, and Cry-negative BMB171 on the colorectal cancer cell line SW480 reported 30-50% of cellular viability except for BMB171. Molecular dynamic simulations performed to identify whether the mutations at positions 553 and 556 were related to the stability and rigidity of the functional tertiary structure (domain III) of the Cry11Aa protein and variant 8 showed the importance of these mutations in specific regions for the toxic activity of Cry11 against A. aegypti. This generates pertinent knowledge for the design of Cry11 proteins and their biotechnological applications in vector-borne disease control and cancer cell lines.

Identification of 7-Ketocholesterol-Modulated Pathways and Sterculic Acid Protective Effect in Retinal Pigmented Epithelium Cells by Using Genome-Wide Transcriptomic Analysis.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. AMD is characterized by the formation of lipidic deposits between the retinal pigment epithelium (RPE) and the choroid called drusen. 7-Ketocholesterol (7KCh), an oxidized-cholesterol derivative, is closely related to AMD as it is one of the main molecules accumulated in drusen. 7KCh induces inflammatory and cytotoxic responses in different cell types, and a better knowledge of the signaling pathways involved in its response would provide a new perspective on the molecular mechanisms that lead to the development of AMD. Furthermore, currently used therapies for AMD are not efficient enough. Sterculic acid (SA) attenuates the 7KCh response in RPE cells and is presented as an alternative to improve these therapies. By using genome-wide transcriptomic analysis in monkey RPE cells, we have provided new insight into 7KCh-induced signaling in RPE cells, as well as the protective capacity of SA. 7KCh modulates the expression of several genes associated with lipid metabolism, endoplasmic reticulum stress, inflammation and cell death and induces a complex response in RPE cells. The addition of SA successfully attenuates the deleterious effect of 7KCh and highlights its potential for the treatment of AMD.

Connection between MHC class II binding and aggregation propensity: The antigenic peptide 10 of Paracoccidioides brasiliensis as a benchmark study.

The aggregation of epitopes that are also able to bind major histocompatibility complex (MHC) alleles raises questions around the potential connection between the formation of epitope aggregates and their affinities to MHC receptors. We first performed a general bioinformatic assessment over a public dataset of MHC class II epitopes, finding that higher experimental binding correlates with higher aggregation-propensity predictors. We then focused on the case of P10, an epitope used as a vaccine candidate against Paracoccidioides brasiliensis that aggregates into amyloid fibrils. We used a computational protocol to design variants of the P10 epitope to study the connection between the binding stabilities towards human MHC class II alleles and their aggregation propensities. The binding of the designed variants was tested experimentally, as well as their aggregation capacity. High-affinity MHC class II binders in vitro were more disposed to aggregate forming amyloid fibrils capable of binding Thioflavin T and congo red, while low affinity MHC class II binders remained soluble or formed rare amorphous aggregates. This study shows a possible connection between the aggregation propensity of an epitope and its affinity for the MHC class II cleft.

NTD Health: an electronic medical record system for neglected tropical diseases / NTD Health: un sistema de historias clínicas electrónicas para enfermedades tropicales desatendidas

INTRODUCTION: The use of technological resources to support processes in health systems has generated robust, interoperable and dynamic platforms. In the case of institutions working with neglected tropical diseases (NTD), there is a need for NTD-specific customizations. OBJECTIVES: To establish a medical records platform, specialized for NTD, which would facilitate the analysis of treatment evolution in patients, as well as generate more accurate data about various clinical aspects. MATERIALS AND METHODS: Here we developed a customized electronic medical record system based on OpenMRS for multiple NTDs. A set of forms and functionalities was developed under the OpenMRS guidelines, using shared community modules. RESULTS: All the customized information was packaged in a distribution called NTD Health. The platform is web-based and can be upgraded and improved by users without technological barriers. CONCLUSIONS: The EMR system can become a useful tool for other institutions to improve their health practices as well as the quality of life for NTD patients, simplifying the customization of healthcare systems able to interoperate with other platforms.

Introducción. El uso de recursos tecnológicos destinados a apoyar procesos en los sistemas de salud ha generado plataformas sólidas, interoperables y dinámicas. En el caso de las instituciones que trabajan con enfermedades tropicales desatendidas, existe la necesidad de personalizaciones específicas en las herramientas de uso médico. Objetivos. Establecer una plataforma para historias clínicas especializada en enfermedades tropicales desatendidas, con el fin de facilitar el análisis de la evolución del tratamiento de los pacientes, además de generar datos más precisos sobre diversos aspectos clínicos. Materiales y métodos. Se compiló un conjunto de requisitos para implementar formularios, conceptos y funcionalidades que permitan incluir enfermedades tropicales desatendidas. Se utilizó una distribución de OpenMRS (versión 2.3) como referencia para construir la plataforma, siguiendo las pautas recomendadas y módulos compartidos por la comunidad. Resultados. Toda la información personalizada se implementó en una plataforma llamada NTD Health, la cual se encuentra almacenada en la web y los usuarios pueden actualizarla y mejorarla sin barreras tecnológicas. Conclusiones. El sistema de historias clínicas electrónicas puede convertirse en una herramienta útil para que otras instituciones mejoren sus prácticas en salud, así como la calidad de vida de los pacientes con enfermedades tropicales desatendidas, simplificando la personalización de los sistemas de salud capaces de interoperar con otras plataformas.

NTD Health: An electronic medical record system for neglected tropical diseases / NTD Health: un sistema de historias clínicas electrónicas para enfermedades tropicales desatendidas

Introduction: The use of technological resources to support processes in health systems has generated robust, interoperable, and dynamic platforms. In the case of institutions working with neglected tropical diseases, there is a need for specific customizations of these diseases. Objectives: To establish a medical record platform specialized in neglected tropical diseases which could facilitate the analysis of treatment evolution in patients, as well as generate more accurate data about various clinical aspects. Materials and methods: A set of requirements to develop state of the art forms, concepts, and functionalities to include neglected tropical diseases were compiled. An OpenMRS distribution (version 2.3) was used as reference to build the platform, following the recommended guidelines and shared-community modules. Results: All the customized information was developed in a platform called NTD Health, which is web-based and can be upgraded and improved by users without technological barriers. Conclusions: The electronic medical record system can become a useful tool for other institutions to improve their health practices as well as the quality of life for neglected tropical disease patients, simplifying the customization of healthcare systems able to interoperate with other platforms.

Introducción. El uso de recursos tecnológicos destinados a apoyar procesos en los sistemas de salud ha generado plataformas sólidas, interoperables y dinámicas. En el caso de las instituciones que trabajan con enfermedades tropicales desatendidas, existe la necesidad de personalizaciones específicas en las herramientas de uso médico. Objetivos. Establecer una plataforma para historias clínicas especializada en enfermedades tropicales desatendidas, con el fin de facilitar el análisis de la evolución del tratamiento de los pacientes, además de generar datos más precisos sobre diversos aspectos clínicos. Materiales y métodos. Se compiló un conjunto de requisitos para implementar formularios, conceptos y funcionalidades que permitan incluir enfermedades tropicales desatendidas. Se utilizó una distribución de OpenMRS (versión 2.3) como referencia para construir la plataforma, siguiendo las pautas recomendadas y módulos compartidos por la comunidad. Resultados. Toda la información personalizada se implementó en una plataforma llamada NTD Health, la cual se encuentra almacenada en la web y los usuarios pueden actualizarla y mejorarla sin barreras tecnológicas. Conclusiones. El sistema de historias clínicas electrónicas puede convertirse en una herramienta útil para que otras instituciones mejoren sus prácticas en salud, así como la calidad de vida de los pacientes con enfermedades tropicales desatendidas, simplificando la personalización de los sistemas de salud capaces de interoperar con otras plataformas.

Protocol for iterative optimization of modified peptides bound to protein targets.

Peptides are commonly used as therapeutic agents. However, they suffer from easy degradation and instability. Replacing natural by non-natural amino acids can avoid these problems, and potentially improve the affinity towards the target protein. Here, we present a computational pipeline to optimize peptides based on adding non-natural amino acids while improving their binding affinity. The workflow is an iterative computational evolution algorithm, inspired by the PARCE protocol, that performs single-point mutations on the peptide sequence using modules from the Rosetta framework. The modifications can be guided based on the structural properties or previous knowledge of the biological system. At each mutation step, the affinity to the protein is estimated by sampling the complex conformations and applying a consensus metric using various open protein-ligand scoring functions. The mutations are accepted based on the score differences, allowing for an iterative optimization of the initial peptide. The sampling/scoring scheme was benchmarked with a set of protein-peptide complexes where experimental affinity values have been reported. In addition, a basic application using a known protein-peptide complex is also provided. The structure- and dynamic-based approach allows users to optimize bound peptides, with the option to personalize the code for further applications. The protocol, called mPARCE, is available at: https://github.com/rochoa85/mPARCE/ .

BILN: A Human-Readable Line Notation for Complex Peptides.

We present an easy, human-readable line notation to describe even complex peptides.

In Silico, In Vitro, and Pharmacokinetic Studies of UBMC-4, a Potential Novel Compound for Treating against Trypanosoma cruzi.

The lack of therapeutic alternatives for the treatment of Chagas disease, a neglected disease, drives the discovery of new drugs with trypanocidal activity. Consequently, we conducted in vitro studies using UBMC-4, a potential Trypanosoma cruzi AKT-like pleckstrin homology (PH) domain inhibitory compound found using bioinformatics tools. The half effective concentration (EC50) on intracellular amastigotes was determined at 1.85 ± 1 µM showing low cytotoxicity (LC50) > 40 µM on human cell lines tested. In order to study the lethal effect caused by the compound on epimastigotes, morphological changes were assessed by scanning and transmission electron microscopy. Progressive alterations such as flagellum inactivation, cell size reduction, nuclear structure alteration, condensation of chromatin towards the nuclear periphery, vacuole formation, and mitochondrial swelling with kinetoplast integrity loss were evidenced. In addition, apoptosis-like markers in T. cruzi were assessed by flow cytometry, demonstrating that the effect of UBMC-4 on T. cruzi AKT-like kinase reduced the tolerance to nutritional stress-triggered, apoptosis-like events, including DNA fragmentation, mitochondrial damage, and loss of plasma membrane integrity. After this, UBMC-4 was formulated for oral administration and pharmacokinetics were analyzed in a mouse model. Finally, upon oral administration of 200 mg/kg in mice, we found that a UBMC-4 plasma concentration remaining in circulation beyond 24 h after administration is well described by the two-compartment model. We conclude that UBMC-4 has an effective trypanocidal activity in vitro at low concentrations and this effect is evident in T. cruzi cell structures. In mice, UBMC-4 was well absorbed and reached plasma concentrations higher than the EC50, showing features that would aid in developing a new drug to treat Chagas disease.

Multiple-Allele MHC Class II Epitope Engineering by a Molecular Dynamics-Based Evolution Protocol.

Epitopes that bind simultaneously to all human alleles of Major Histocompatibility Complex class II (MHC II) are considered one of the key factors for the development of improved vaccines and cancer immunotherapies. To engineer MHC II multiple-allele binders, we developed a protocol called PanMHC-PARCE, based on the unsupervised optimization of the epitope sequence by single-point mutations, parallel explicit-solvent molecular dynamics simulations and scoring of the MHC II-epitope complexes. The key idea is accepting mutations that not only improve the affinity but also reduce the affinity gap between the alleles. We applied this methodology to enhance a Plasmodium vivax epitope for multiple-allele binding. In vitro rate-binding assays showed that four engineered peptides were able to bind with improved affinity toward multiple human MHC II alleles. Moreover, we demonstrated that mice immunized with the peptides exhibited interferon-gamma cellular immune response. Overall, the method enables the engineering of peptides with improved binding properties that can be used for the generation of new immunotherapies.

Computational Evolution Protocol for Peptide Design.

Computational peptide design is useful for therapeutics, diagnostics, and vaccine development. To select the most promising peptide candidates, the key is describing accurately the peptide-target interactions at the molecular level. We here review a computational peptide design protocol whose key feature is the use of all-atom explicit solvent molecular dynamics for describing the different peptide-target complexes explored during the optimization. We describe the milestones behind the development of this protocol, which is now implemented in an open-source code called PARCE. We provide a basic tutorial to run the code for an antibody fragment design example. Finally, we describe three additional applications of the method to design peptides for different targets, illustrating the broad scope of the proposed approach.

In vitro and in silico study of an exclusive insertion in the nicotinamide/nicotinate mononucleotide adenylyltransferase from Leishmania braziliensis.

The intracellular parasite Leishmania braziliensis is the causal agent of cutaneous and mucocutaneous leishmaniasis, a group of endemic diseases in tropical regions, including Latin America. New therapeutic targets are required to inhibit the pathogen without affecting the host. The enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18) is a potential target, since it catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), which is an essential metabolite in multiple cellular processes. In this work, we produced and evaluated the catalytic activity of the recombinant protein 6HisΔ241-249LbNMNAT to study the functional relevance of the exclusive insertion present in the enzyme of L. braziliensis (LbNMNAT), but absent in the primary structure of human NMNATs. Our results indicate that the 241-249 insertion constitutes a structural element that connects the protein structure Rossmann topology with the carboxyl-terminal domain of the enzyme. The removal of this region drastically decreases the solubility, and enzymatic activity of the recombinant, causing its inactivation. Molecular dynamics simulations were carried out with the wild-type and truncated enzymes to verify additional changes in their stability, which indicated a better stability in the wild-type protein. These findings constitute an initial step to identify a new inhibition mechanism for the development of focused pharmacological strategies on exclusive insertions from the LbNMNAT protein.

Prediction of potential cysteine synthase inhibitors of Leishmania braziliensis and Leishmania major parasites by computational screening.

Leishmaniasis is a neglected tropical disease considered a public health problem that requires innovative strategies for its chemotherapeutic control. In the present investigation, a molecular docking approach was carried out using the protein cysteine synthase (CS) of Leishmania braziliensis (CSLb) and Leishmania major (CSLm) parasites to identify new compounds as potential candidates for the development of selective leishmaniasis therapy. CS protein sequence similarity, active site, structural modeling, molecular docking, and ADMET properties of compounds were analyzed using bioinformatics tools. Molecular docking analyses identified 1000 ligands with highly promising binding affinity scores for both CS proteins. A total of 182 compounds for CSLb and 173 for CSLm were selected for more detailed characterization based on the binding energy and frequency values and ADMET properties. Based on Principal Component Analysis (PCA) and K-means clusterization for both CS proteins, we classified compounds into 5 clusters for CSLb and 7 for CSLm, thus providing an excellent starting point for verification of enzyme inhibition in in vitro studies. We found the ZINC16524774 compound predicted to have a high affinity and stability for both CSLb and CSLm proteins, which was also evaluated through molecular dynamics simulations. Compounds within each of the five clusters also displayed pharmacological and structural properties that make them attractive drug candidates for the development of selective cutaneous leishmaniasis chemotherapy.

Predictive Biomarkers of Age-Related Macular Degeneration Response to Anti-VEGF Treatment.

Age-related macular degeneration (AMD) is an incurable disease associated with aging that destroys sharp and central vision. Increasing evidence implicates both systemic and local inflammation in the pathogenesis of AMD. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is currently the first-line therapy for choroidal neovascularization in AMD patients. However, a high number of patients do not show satisfactory responses to anti-VEGF treatment after three injections. Predictive treatment response models are one of the most powerful tools for personalized medicine. Therefore, the application of these models is very helpful to predict the optimal treatment for an early application on each patient. We analyzed the transcriptome of peripheral blood mononuclear cells (PBMCs) from AMD patients before treatment to identify biomarkers of response to ranibizumab. A classification model comprised of four mRNAs and one miRNA isolated from PBMCs was able to predict the response to ranibizumab with high accuracy (Area Under the Curve of the Receiver Operating Characteristic curve = 0.968), before treatment. We consider that our classification model, based on mRNA and miRNA from PBMCs allows a robust prediction of patients with insufficient response to anti-VEGF treatment. In addition, it could be used in combination with other methods, such as specific baseline characteristics, to identify patients with poor response to anti-VEGF treatment to establish patient-specific treatment plans at the first visit.

Sterculic Acid Alters Adhesion Molecules Expression and Extracellular Matrix Compounds to Regulate Migration of Lung Cancer Cells.

Sterculic acid (SA) is a cyclopropenoid fatty acid isolated from Sterculia foetida seeds. This molecule is a well-known inhibitor of SCD1 enzyme, also known as ∆9-desaturase, which main function is related to lipid metabolism. However, recent studies have demonstrated that it also modifies many other pathways and the underlying gene expression. SCD overexpression, or up-regulated activity, has been associated with tumor aggressiveness and poor prognosis in many cancer types. Scd1 down-regulation, with different inhibitors or molecular strategies, reduces tumor cell survival and cell proliferation, as well as the chemoresistance associated with cancer stem cell presence. However, SA effects over cancer cell migration and extracellular matrix or adhesion molecules have not been described in cancer cells up to now. We used different migration assays and qPCR gene expression analysis to evaluate the effects of SA treatment in cancer cells. The results reveal that SA induces tumoral cell death at high doses, but we also observed that lower SA-treatments induce cell adhesion-migration capacity reduction as a result of modifications in the expression of genes related to integrins and extracellular matrix compounds. Overall, the functional and transcriptomic findings suggest that SA could represent a new inhibitor activity of epithelial to mesenchymal transition.

dockECR: Open consensus docking and ranking protocol for virtual screening of small molecules.

The development of open computational pipelines to accelerate the discovery of treatments for emerging diseases allows finding novel solutions in shorter periods of time. Consensus molecular docking is one of these approaches, and its main purpose is to increase the detection of real actives within virtual screening campaigns. Here we present dockECR, an open consensus docking and ranking protocol that implements the exponential consensus ranking method to prioritize molecular candidates. The protocol uses four open source molecular docking programs: AutoDock Vina, Smina, LeDock and rDock, to rank the molecules. In addition, we introduce a scoring strategy based on the average RMSD obtained from comparing the best poses from each single program to complement the consensus ranking with information about the predicted poses. The protocol was benchmarked using 15 relevant protein targets with known actives and decoys, and applied using the main protease of the SARS-CoV-2 virus. For the application, different crystal structures of the protease, and frames obtained from molecular dynamics simulations were used to dock a library of 79 molecules derived from previously co-crystallized fragments. The ranking obtained with dockECR was used to prioritize eight candidates, which were evaluated in terms of the interactions generated with key residues from the protease. The protocol can be implemented in any virtual screening campaign involving proteins as molecular targets. The dockECR code is publicly available at: https://github.com/rochoa85/dockECR.

Identification of Potential Kinase Inhibitors within the PI3K/AKT Pathway of Leishmania Species.

Leishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases' participation in protein-protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasite.

Application of ensemble pharmacophore-based virtual screening to the discovery of novel antimitotic tubulin inhibitors.

Tubulin is a well-validated target for herbicides, fungicides, anti-parasitic, and anti-tumor drugs. Many of the non-cancer tubulin drugs bind to its colchicine site but no colchicine-site anticancer drug is available. The colchicine site is composed of three interconnected sub-pockets that fit their ligands and modify others' preference, making the design of molecular hybrids (that bind to more than one sub-pocket) a difficult task. Taking advantage of the more than eighty published X-ray structures of tubulin in complex with ligands bound to the colchicine site, we generated an ensemble of pharmacophore representations that flexibly sample the interactional space between the ligands and target. We searched the ZINC database for scaffolds able to fit several of the subpockets, such as tetrazoles, sulfonamides and diarylmethanes, selected roughly ~8000 compounds with favorable predicted properties. A Flexi-pharma virtual screening, based on ensemble pharmacophore, was performed by two different methodologies. Combining the scaffolds that best fit the ensemble pharmacophore-representation, we designed a new family of ligands, resulting in a novel tubulin modulator. We synthesized tetrazole 5 and tested it as a tubulin inhibitor in vitro. In good agreement with the design principles, it demonstrated micromolar activity against in vitro tubulin polymerization and nanomolar anti-proliferative effect against human epithelioid carcinoma HeLa cells through microtubule disruption, as shown by immunofluorescence confocal microscopy. The integrative methodology succedes in the design of new scaffolds for flexible proteins with structural coupling between pockets, thus expanding the way in which computational methods can be used as significant tools in the drug design process.