Small molecules, big targets: drug discovery faces the protein-protein interaction challenge.

Protein-protein interactions (PPIs) are of pivotal importance in the regulation of biological systems and are consequently implicated in the development of disease states. Recent work has begun to show that, with the right tools, certain classes of PPI can yield to the efforts of medicinal chemists to develop inhibitors, and the first PPI inhibitors have reached clinical development. In this Review, we describe the research leading to these breakthroughs and highlight the existence of groups of structurally related PPIs within the PPI target class. For each of these groups, we use examples of successful discovery efforts to illustrate the research strategies that have proved most useful.JS, DES and ARB thank the Wellcome Trust for funding.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nrd.2016.2

Understanding and Drugging the Bcl-2 Transmembrane Interactome for Tumor Treatment

[ES] La familia de proteínas Bcl-2 regula la apoptosis a través de una compleja red de interacciones. Las células tumorales suelen presentar mutaciones que afectan a su expresión o sus interacciones para mejorar la progresión tumoral. Además, alteraciones en su regulación también promueven la migración de células cancerígenas, la invasión y la metástasis. Para llevar a cabo sus funciones, las proteínas Bcl 2 interaccionan entre sí tanto en el citoplasma como en las membranas intracelulares. Los equilibrios de interacción de los dominios Bcl citosólicos se han investigado ampliamente y recientemente, se han propuesto como dianas terapéuticas. Sin embargo, el interactoma de los dominios transmembrana (TMD, del inglés transmembrane domains) sigue siendo poco conocido. Por ello, un conocimiento profundo de la biología de las proteínas Bcl-2 es necesario para explotar eficientemente sus superficies de unión en el tratamiento del cáncer. Para llevar a cabo este objetivo, nos hemos centrado en tres áreas: 1. La comprensión detallada de la contribución del TMD de Mcl-1 a su interactoma en membrana y su función. 2. El descubrimiento de nuevos inhibidores de Mcl-1 que actúen sobre su TMD y que permitan desarrollar una clase de drogas anticancerígenas aún por explorar. 3. La caracterización molecular de mutaciones relacionadas con el cáncer descritas en los TMD de Bcl-2 y Bcl-xL y sus implicaciones en la supervivencia de las células tumorales. La proteína antiapoptótica Mcl-1 inhibe a los miembros proapoptóticos Bak, Bax, Bok, Noxa, etc. Aunque se ha estudiado en detalle su actividad promoviendo la supervivencia celular, el mecanismo molecular por el cuál previene la apoptosis mediada por Bok aún no está claro. Además, el conocimiento de las actividades de Mcl-1, descritas hasta ahora, se basa exclusivamente en las estructuras resueltas de las regiones solubles en agua y en estudios centrados en los dominios citosólicos. Por primera vez, hemos demostrado la relevancia del TMD de Mcl-1 en su equilibrio de interacción. En este trabajo describimos su capacidad específica para homo- y hetero-oligomerizar con el TMD de Bok. También ponemos de manifiesto la influencia de estas interacciones en la modulación de apoptosis y resaltamos la relevancia clínica de los mutantes del TMD de Mcl-1 identificados en pacientes con cáncer. Muchos tumores hematológicos y sólidos sobre-expresan Mcl-1 como mecanismo para adquirir quimiorresistencia. Se han desarrollado miméticos de BH3 específicos para modular su actividad antiapoptótica en células cancerosas. Sin embargo, aún no disponemos de datos científicos que informen sobre su toxicidad y eficacia en humanos. En este trabajo, proponemos la novedosa interacción de los TMDs de Mcl-1 y Bok como un nuevo sitio de acción de fármacos quimioterapéuticos. Hemos identificado tres inhibidores de esta interacción con características que los hacen prometedores candidatos para el desarrollo farmacéutico, así como buenas herramientas moleculares para estudiar la interacción de los TMDs de Mcl-1 y Bok. Para modular la apoptosis, las células tumorales también presentan versiones mutadas de las proteínas antiapoptóticas Bcl-2 y Bcl-xL. En nuestro conocimiento, este es el primer estudio que analiza mutaciones somáticas de sus TMDs. Nuestro trabajo demuestra cómo estas mutaciones alteran el equilibrio en membrana de las proteínas. Además, nuestros resultados explican la influencia que algunos mutantes somáticos ejercen en la regulación de la apoptosis. En general, los resultados científicos que aparecen en esta tesis resaltan el papel de los Bcl TMDs en el interactoma de las proteínas Bcl-2. Estos hallazgos corroboran que las interacciones laterales entre los TMDs son específicas y contribuyen activamente a la funcionalidad de la proteína. Por lo tanto, comprender los Bcl TMDs puede proporcionar nuevos conocimientos sobre la biología de las proteínas Bcl.[CA] La família de proteïnes Bcl-2 regula l'apoptosi a través d'una complexa xarxa d'interaccions. Les cèl·lules tumorals solen presentar mutacions que afecten la seua expressió o les seues interaccions per a millorar la progressió tumoral. A més, alteracions en la seua regulació també promouen la migració de cèl·lules cancerígenes, la invasió i la metàstasi. Per a dur a terme les seues funcions, les proteïnes Bcl-2 interaccionen entre si tant en el citoplasma com en les membranes intracel·lulars. Els equilibris d'interacció dels dominis Bcl citosòlics s'han investigat àmpliament i recentment, s'han proposat com a dianes terapèutiques. No obstant això, l'interactoma dels dominis transmembrana (TMD, de l'anglés transmembrane domains) continua sent poc conegut. Per això, un coneixement profund de la biologia de les proteïnes Bcl-2 és necessari per a explotar eficientment les seues superfícies d'unió en el tractament del càncer. Per a dur a terme aquest objectiu, ens hem centrat en tres àrees: 1. La comprensió detallada de la contribució del TMD de Mcl-1 al seu interactoma en membrana i la seua funció. 2. El descobriment de nous inhibidors de Mcl-1 que actuen sobre el seu TMD i que permeten desenvolupar una classe de drogues anticanceroses encara per explorar. 3. La caracterització molecular de mutacions relacionades amb el càncer descrites en els TMD de Bcl-2 i Bcl-xL i les seues implicacions en la supervivència de les cèl·lules tumorals. La proteïna anti apoptòtica Mcl-1 inhibeix als membres pro apoptòtics Bak, Bax, Bok, Noxa, etc. Encara que s'ha estudiat detalladament la seua activitat promovent la supervivència cel·lular, el mecanisme molecular pel qual prevé l'apoptosi mediada per Bok encara no és clar. A més, el coneixement de les activitats de Mcl-1, descrites fins ara, es basa exclusivament en les estructures resoltes solubles en aigua i en estudis centrats en els dominis externs a la membrana. Per primera vegada, hem demostrat la rellevància del TMD de Mcl-1 el seu equilibri d'interacció. En aquest treball descrivim la seua capacitat específica per a unir-se amb si mateix i per a hetero-oligomeritzar amb el TMD de Bok. També expliquem la influència d'aquestes interaccions en l'apoptosi i ressaltem la rellevància clínica dels mutants del TMD de Mcl-1 identificats en pacients amb càncer. Molts tumors hematològics i sòlids sobre-expressen Mcl-1 com un mecanisme per a adquirir quimioresistència. S'han desenvolupat mimètics de BH3 específics per a modular la seua activitat anti apoptòtica en cèl·lules canceroses. No obstant això, encara no disposem de dades científiques que informen sobre la seua toxicitat i eficàcia en humans. Per això, proposem la nova interacció dels TMDs de Mcl-1 i Bok com un lloc d'actuació de fàrmacs quimioterapèutiques. Hem identificat tres inhibidors d'aquesta interacció amb característiques que els fan prometedors candidats per al desenvolupament farmacèutic, així com bones eines moleculars per a estudiar la interacció dels TMDs de Mcl-1 i Bok. Per a modular l'apoptosi, les cèl·lules tumorals també presenten versions mutades de les proteïnes anti apoptòtiques Bcl-2 i Bcl-xL. En el nostre coneixement, aquest és el primer estudi que analitza mutacions somàtiques de les seues TMDs. El nostre treball demostra com aquestes mutacions alteren l'equilibri en membrana de les proteïnes. A més, els nostres resultats expliquen la influència que alguns mutants somàtics exerceixen en la regulació de l'apoptosi. En general, els resultats científics que apareixen en aquesta tesi ressalten el paper dels Bcl TMDs en l'interactoma de les proteïnes Bcl-2. Aquestes troballes corroboren que les interaccions laterals entre els TMDs són específiques de la seqüència i contribueixen activament a la funcionalitat de la proteïna. Per tant, comprendre els Bcl TMDs pot proporcionar nous coneixements sobre la biologia de les proteïnes Bcl[EN] The family of the Bcl-2 proteins modulates the apoptotic pathway by a complex network of interactions. Tumor cells frequently present mutations that affect Bcl-2 proteins expression or interactions to enhance cancer progression. Dysregulation of these proteins also promotes cancer cell migration, invasion, and metastasis. To execute their functions, Bcl-2 proteins interact in both the cytosol and intracellular membranes. Binding equilibria of Bcl extramembrane domains has been largely investigated and recently proposed as chemotherapeutic targets. However, the interactome of transmembrane domains (TMDs) remains poorly understood. In this scenario, a deep knowledge of the biology of Bcl-2 proteins is needed to exploit efficiently their binding surfaces for cancer treatment. To address this aim, our research focuses on three areas: 1. The detailed comprehension of the TMD contribution to both the Mcl-1 membrane interactome and protein functionality. 2. The discovery of new Mcl-1 inhibitors that target the transmembrane surface to develop a class of anticancer drugs currently unexplored. 3. The molecular characterization of cancer-related mutations within the Bcl-2 and Bcl-xL TMDs and their implications for the survival of cancer cells. Antiapoptotic Mcl-1 protein inhibits the proapoptotic members Bak, Bax, Bok, and Noxa, among others. Although its prosurvival activity has been well studied, the molecular mechanism to prevent Bok-mediated apoptosis remains unclear. Furthermore, understanding of Mcl-1 activities described to date is only based on water-soluble structures and studies focused on extramembrane domains. For the first time, we uncover the relevance of the Mcl-1 TMD in the interaction equilibria of the protein. In the present work, we describe its specific capacity to self-associate and hetero-oligomerize with the Bok TMD. We also explain the influence of these interactions in the apoptotic pathway and highlight the clinical relevance of Mcl-1 TMD mutants identified in tumor patients. Many hematological and solid malignancies overexpress Mcl-1 as an acquired chemoresistance mechanism. To modulate its antiapoptotic activity in cancer cells, specific BH3 mimetics have been developed; however, there is no scientific data yet regarding human toxicity and efficacy. In this work, we propose the novel Mcl-1 and Bok TMDs interaction interface as a drugging site in the development of chemotherapeutics. We identify three potential inhibitors of such molecular interface with promising features to become both drug candidates for pharmaceutical development and research toosl for the molecular study of the Mcl-1 and Bok TMDs interaction. To take advantage of apoptosis modulation, tumor cells also present mutated versions of the antiapoptotic members Bcl-2 and Bcl-xL. To our knowledge, this is the first study that analyzes patient-derived mutations within Bcl-2 and Bcl-xL TMDs and demonstrates how said mutations alter the membrane equilibria of these proteins. The results presented here also explain the functional influence of some somatic mutants in apoptosis regulation. Overall, the scientific results exhibited in this Thesis highlight the role of Bcl TMDs in the interactome of Bcl-2 proteins. These findings corroborate that lateral interactions between TMDs are sequence-specific and actively contribute to protein functionality. Therefore, understanding of Bcl transmembrane segments may provide new insights into the biology of Bcl 2 proteins for their pharmaceutical modulation in antitumoral therapy.The student has been granted with a PhD fellowship and a short-term fellowship from the Generalitat Valenciana (Subvenciones para la contratación de personal investigador de carácter predoctoral, 2016-2019, and Grant for predoctoral stays out of the Comunitat Valenciana, 2019). This work has been supported by the Spanish Ministry of Economy and Competitiveness (projects SAF2014-52614-R and SAF2017-84689-RLucendo Gutiérrez, E. (2020). Understanding and Drugging the Bcl-2 Transmembrane Interactome for Tumor Treatment [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/155914TESI

IN SILICO DOCKING STUDIES OF GALLIC ACID STRUCTURAL ANALOGUES AS BCL-XL INHIBITOR IN CANCER

Objective: Apoptosis, or programed cell death, forms an important part of the cellular regulation machinery. The Bcl-2 protein family, comprisingproapoptotic and antiapoptotic members, forms an important part of the cells internal apoptotic pathway. Overexpression of the antiapoptoticmembers of the family in a number of cancer cell lines renders them immune to apoptosis and the ability to survive under conditions of cellular stress.Inhibition of the antiapoptotic members of the Bcl-2 family like Bcl-XL is, therefore, an interesting target for the development of anticancer therapy.Methods: The structure of antiapoptotic Bcl-XL receptor (1YSG) was taken from PDB database. The 23-dimensional structure of gallic acid analogswas designed. The Lipinski properties of gallic acid analogs were calculated using molsoft. Docking studies have been carried out through Autodock4.0 software.Results: Molecular docking analysis with gallic acid and their structural analogs showed propyl gallate, benzyl gallate, diallyl gallate, phenyl ethylgallate, and allyl gallate are more interactive and binding strongly than gallic acid at active site of Bcl-XL.Conclusion: Further these five compounds should be considered as potential candidates for Bcl-XL inhibitor.Keywords: Apoptosis, Bcl-2, Antiapoptotic Bcl-XL receptor, Gallic acid, Docking studies

Low molecular weight compounds from mushrooms as potential Bcl-2 inhibitors: docking and virtual screening studies

Mestrado com dupla diplomação com o Institut Superieur de Biotechnologie de MonastirMushrooms have the ability to promote apoptosis in tumor cell lines, but the mechanism of action is not quite well understood. Inhibition of the interaction between Bcl-2 and pro-apoptotic proteins could be an important step that leads to apoptosis. Therefore, the discovery of compounds with the ability to inhibit Bcl-2 is an ongoing research topic in drug discovery. In this study, we started by analyzing Bcl-2 experimental structures that are currently available in Protein Data Bank database. After analysis of the more relevant Bcl-2 structures, 4 were finally selected. An analysis of the best docking methodology was then performed using a cross-docking and re-docking approach while testing 2 docking softwares: AutoDock 4 and AutoDock Vina. Autodock4 provided the best docking results and was selected to perform a virtual screening study applied to a dataset of 40 Low Molecular Weight (LMW) compounds present in mushrooms, using the selected Bcl-2 structures as target. Results suggest that steroid are the more promising family, among the analyzed compounds, and may have the ability to interact with Bcl-2 and this way promoting tumor apoptosis. The steroids that presented lowest estimated binding energy (ΔG) were: Ganodermanondiol, Cerevisterol, Ganoderic Acid X and Lucidenic Lactone; with estimated ΔG values between -8,45 and -8,23 Kcal/mol. A detailed analysis of the docked conformation of these 4 top ranked LMW compounds was also performed and illustrates a plausible interaction between the 4 top raked steroids and Bcl-2, thus substantiating the accuracy of the predicted docked poses. Therefore, tumoral apoptosis promoted by mushroom might be related to Bcl-2 inhibition mediated by steroid family of compounds.Os cogumelos apresentam a capacidade de promover a apoptose em linhas células tumorais, No entanto o seu mecanismo de ação não é completamente conhecido. A inibição da interação entre Bcl-2 e proteínas pro-apoptóticas pode ser um passo importante na iniciação do processo de apoptose tumoral. Por essa razão, a descoberta de compostos que inibam a proteína Bcl-2 é uma área importante na descoberta de novos fármacos antitumorais. Neste estudo, começou-se por analisar as estruturas experimentais de Bcl-2 atualmente presentes na base de estruturas Protein Data Bank. Após análise das estruturas de Bcl-2 mais relevantes, 4 foram escolhidas. Um estudo de “cross-docking” e “re-docking” foi então realizado para escolher a metodologia de “docking” mais adequada. Testaram-se 2 softwares, o AutoDock 4 e o AutoDock Vina, e verificou-se que o AutoDock 4 apresentava melhores resultados, tendo sido o selecionado para realizar os ensaios de “screening” virtual dos 40 compostos de baixo peso molecular presentes em cogumelos, utilizando as 4 estruturas selecionadas. Os resultados obtidos sugerem que os esteroides são a família de compostos mais prometedores de entre as famílias de compostos estudadas. Os esteroides que apresentaram valores de energia de ligação (ΔG) mais baixos foram: Ganodermanondiol, Cerevisterol, Ácido Ganoderico X and Lactona Lucidénica, com valores de ΔG estimado entre -8,45 e -8,23 Kcal/mol. Uma análise detalhada da conformação de ligação foi também realizada dos 4 melhores compostos de baixo peso molecular melhor classificados. Esta análise demonstra um modo de interação plausível entre os compostos e a estrutura da Bcl-2, consubstanciando a eficácia dos resultados obtidos por “docking”. Conclui-se que o processo inibição de apoptose tumoral observada em cogumelos pode estar relacionado com a inibição da Bcl-2 por esteroides presentes nos cogumelos

Selective Small Molecule Targeting of Anti-Apoptotic MCL-1

BCL-2 family proteins are key regulators of the mitochondrial apoptotic pathway in health and disease. Anti-apoptotic members such as BCL-2, $BCL-X_L$, and MCL-1 have been implicated in the initiation, progression, and chemoresistance of human cancer. Small molecules and peptides have successfully targeted the anti-apoptotic BCL-2/$BCL-X_L$ groove that binds and sequesters pro-apoptotic BH3 death helices. Such compounds induce tumor cell apoptosis and are being advanced in clinical trials as promising next-generation cancer therapeutics. Notably, selective antagonists such as ABT-737 are highly effective at inducing apoptosis in BCL-2/$BCL-X_L$-dependent cancers but are rendered inactive by overexpression of MCL-1, a formidable chemoresistance protein that lies outside the molecule's binding spectrum. By screening a library of stabilized alpha-helices of BCL-2 domains (SAHBs), we previously discovered that the MCL-1 BH3 helix is itself a potent and exclusive MCL-1 inhibitor. Here, we deployed this chemically-constrained peptidic inhibitor of MCL-1, MCL-1 SAHB, in a competitive binding screen to identify selective small molecule inhibitors of MCL-1. Rigorous in vitro binding and functional assays were used to validate the compounds and their mechanisms of action, and most notably, MCL-1 inhibitor molecule 1 (MIM1) displayed exquisite selectivity in these assays. NMR analysis documented that MIM1 engages the canonical BH3-binding pocket of MCL-1. Importantly, MIM1 selectively triggers caspase 3/7 activation and apoptosis of a cancer cell line that is dependent on induced overexpression of MCL-1 but showed no activity in the isogenic cell line that is driven instead by overexpressed $BCL-X_L$. Thus, a selective stapled peptide inhibitor of MCL-1 was successfully applied to identify a high fidelity small molecule inhibitor of MCL-1 that exhibits anti-cancer activity in the specific context of MCL-1 dependence

THE SMALL GTPASE RAC1 IS A NOVEL BINDING PARTNER OF BCL-2 AND STABILIZES ITS ANTI-APOPTOTIC ACTIVITY

Ph.DDOCTOR OF PHILOSOPH

Development of Methods for the Discovery of Small Molecule Biological Probes

Advances in combinatorial chemistry have facilitated the production of large chemical libraries that can be used as tools to discover biological probes and therapeutics. High-throughput screening (HTS) strategies have emerged as the standard method to assess the biological activity of small molecules. These screens involve the individual analysis of each small molecule in multi-well plates, often requiring expensive automated methods and development of robust assays that may not translate to physiologically relevant contexts. This problem of evaluating large numbers of reagents in physiologically relevant cell and animal models has been addressed for genetic reagents such as RNAi, CRISPR, and cDNA by creating barcoded retroviral libraries that can be used to infect target cells in culture or in animal models. Using these tools, effective reagents can be selected and decoded using a rapid and inexpensive procedure compared to testing of individual reagents one at a time in an arrayed fashion. In order to more efficiently analyze small molecules, a pooled approach would similarly be useful. This dissertation describes the studies towards developing a pooled screening strategy for small molecules in cellular contexts. Through an initial screen, we set to phenotypically profile small molecule biological activity in a pooled fashion, while simultaneously gain insight about an individual, active molecule’s mechanism of action. I first describe the design of the pooled screen and define the goals necessary for successful application. Next, I outline the steps taken and challenges encountered during the invention of each component of the technology. Finally, I discuss a computational, target-based approach to design small molecules appropriate for future applications of the new screening technology

Development and Characterization of Novel Mcl-1 Inhibitors for Treatment of Cancer.

Myeloid cell leukemia-1 (Mcl-1) is a potent anti-apoptotic protein, member of the anti-apoptotic Bcl-2 family. Overexpression of Mcl-1 is associated with high tumor grade, resistance to chemotherapy, and poor prognosis in many types of cancers. Thus, Mcl-1 is emerging as a critical survival factor in a broad range of human cancers and represents an attractive molecular target for the development of a new class of cancer therapy. Applying an integrated screening strategy through combining high throughput and virtual screenings, multiple hit compounds with structural diversity were validated as Mcl-1 inhibitors using biochemical and biophysical methods. Based on the confirmed hit molecule and analyzing structure activity relationship (SAR) together with computational docking predicted binding poses supported by HSQC NMR studies, we have designed and optimized a novel class of selective small-molecule inhibitors of Mcl-1 using a 2,4,5 substituted benzoic acid as a scaffold. Several co-crystal structures of this class of inhibitors in complex with Mcl-1 have provided a basis for their further optimization, which ultimately led to the discovery of nanomolar potent and selective ligands that bind to the BH3 hydrophobic groove of the Mcl-1 protein. Mechanistic studies performed in genetically engineered cell lines revealed that our inhibitors have on-target activity and induce Bax/Bak dependent apoptosis; selectively antagonizing Mcl-1 function leading to the induction of the hallmarks of apoptosis. Using functional BH3 profiling assay, we identified heterogeneous dependency on Bcl-2 family members for survival in hematologic malignancies, as well as in solid human cancers. The mitochondrial response to selective Mcl-1 BH3 peptides (Noxa and MS1) predicted the in vitro sensitivity to Mcl-1 inhibitors of several cell lines found to be Mcl-1 dependent, including multiple myeloma cell line H929. 483LM, one of the most potent developed Mcl-1 inhibitors, inhibited the cell growth and induced mechanism-based apoptotic cell death in the H929 cells. Intraperitoneal treatment of the H929 cancer xenograft model with 483LM led to significant dose-dependent tumor regression. Collectively, our data demonstrates that the new class of Mcl-1 inhibitors has promising in vitro and in vivo efficacy, warranting further development toward clinical use in the treatment of human cancers.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135852/1/asmady_1.pdfDescription of asmady_1.pdf : Restricted to UM users only