Enhancing the fight against malaria : from genome to structure and activity of a G-protein coupled receptor from the mosquito, Anopheles Gambiae

Includes abstract.Includes bibliographical references (leaves 183-184).G-proton coupled receptors (GPCRs) are excellent drug targets that occupy a central position in the physiology of insects and are involved in transmission of signal from the extracellular to the intracellular side of the cell. Adipokinetic hormone receptors (AKHRs) are GPCRs that mediate physiological functions of the neurohormones, adipokinetic hormones (AKHs) that regulate mobilisation of energy reserves during mosquito flight. Ligand binding to GPCRs depends on the three dimensional (3D) structures of the receptors but to date no crystal structures of insect GPCRs are available. This work focused on building molecular models of AKHR from the genome of the malaria mosquito, identifying its binding site and studying the conformational and structural changes during molecular dynamics of the active and inactive receptor

Molecular Simulation For Neurodegenerative Diseases: From Modelling Alpha-Synuclein In Aqueous Solutions To Drug Delivery Systems

Using computer simulations, we developed a better understanding of some key chemical steps of alpha-synuclein aggregation as well as amantadine for drug delivery systems. The aggregation and fibrillation of alpha-synuclein, a brain protein of 140 residues, has been linked to several neurodegenerative diseases, including Parkinson\u27s and Alzheimer\u27s diseases. Using molecular dynamics, alpha-synuclein, at various temperatures of 293 K, 310 K, 323 K and 348 K, was shown to undergo rapid conformational change. The non-amyloid- β component (NAC) of alpha-synuclein that is not sterically hindered is suspected to undergo aggregation. Amantadine has been shown to help regulate the symptoms of Parkinson\u27s disease, and single-walled carbon nanotubes (SWCNs) could be a potential drug carrier. Adsorption of amantadine within SWCNs, at differing diameters of 10.9 Å, 16.3 Å, 21.7 Å and 27.1 Å, was studied using Expanded Wang-Landau (EWL) simulations. It was shown that increasing the diameters of the SWCNs increased amantadine loading - monolayers formed for the two smaller SWCNs while bilayers formed for the larger. The diffusion process was studied through molecular dynamics. For the larger two SWCNs, there was a dramatic decrease in the rate of self-diffusion with respect to increasing amantadine loading followed by a moderate decrease. This was attributed to the transition from a monolayer to a bilayer structure

A single AKH neuropeptide activating three different fly AKH-receptors: an insecticide study via computational methods

Flies are a widely distributed pest insect that poses a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis all have the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extracellular binding site of a G protein-coupled receptor causes its activation. In this thesis, the structure of Phote-HrTH in SDS micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be reasonably rigid, with an S 2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, Drosophila melanogaster, the flesh fly, Sarcophaga crassipalpis, and the oriental fruit fly, Bactrocera dorsalis were used to construct two models for each receptor: Drome-AKHR, Sarcr-AKHR, and Bacdo-AKHR. It is proposed that these two models represent the active and inactive state of the receptor. The models based on the crystal structure of the β-2 adrenergic receptor were found to bind Phote-HrTH with a predicted binding free energy of –107 kJ mol–1 for Drome-AKHR, –102 kJ mol–1 for Sarcr-AKHR and –102 kJ mol–1 for Bacdo-AKHR. Under molecular dynamics simulation, in a POPC membrane, the β-2AR receptor-like complexes transformed to rhodopsin-like. The identification and characterisation of the ligand-binding site of each receptor provide novel information on ligand-receptor interactions, which could lead to the development of species-specific control substances to use discriminately against these pest flies

Computational analysis of membrane transporters and their substrates

In this thesis, we developed, implemented and applied bioinformatics tools/techniques in three projects that aim at characterising functional properties of membrane transport systems as well as their interactions with substrates and non-substrates. In the first project, we developed a novel method for MdfA sub- strate classification. MdfA is a multidrug membrane transporter of E. coli, which is responsible for recognising and transporting a wide spectrum of substrates with unrelated properties. Unlike other conventional methods that utilised general features such as sequence derived information, molecular descriptors, etc. , the new method incorporates protein-ligand structural interactions and potential energy information derived from molecular dynamics simulations. However, the method still encountered difficulties with the structural similarity problem between substrates and non-substrates. The new method achieved a decent performance with 73.12% of classification accuracy. Regardless, this is the first method that considers protein- ligand interactions in a classification problem related to membrane transport. In the next project, we analysed the proteomics data from Sec61α and TRAP silencing experiments to reveal and characterise TRAP substrates. TRAP is an assisting component of the translocon complex, which is responsible for protein translocation across the membrane of the endoplasmic reticulum. We successfully identified a set of TRAP dependent proteins from mass spectrometry proteomics data. Furthermore, our analysis revealed that the signal peptides of TRAP substrates showed a low hydrophobicity tendency as well as significantly increased glycine and proline content. We propose that TRAP may be responsible for helping those proteins carrying signal peptides with high glycine-proline content and low hydrophobicity to migrate easily through the Sec61α channel. In the last project, we applied molecular docking to investigate the binding modes of several eeyarestatin compounds (ES1, ES24, ES35 and ES47) to a structural homology model of human Sec61α protein. The Sec61α channel is not only responsible for protein translocation but also promotes Ca2+ leakage. Based on the docking results, we found that the energetically most favourable binding positions of ES1 and ES24 are located in between the H2 and H7 helices, which are the “doors” of the lateral gate. Hence, they are likely to hamper the gate function, keeping it open upon binding. Therefore, we postulated that ES1 and ES24 can be potential “gate blockers” which promote Ca2+ leakage via Sec61α. These findings are consistent with the results from calcium imaging experiments which were conducted by our colleagues.In den vergangenen Jahren haben sich rechnerische Technologien sowie die Entwicklung von anspruchsvollen Algorithmen und Software schnell entwickelt. Diese technologischen Fortschritte spielen einen entscheidende Rolle für die bioinformatische Forschung, da die biologischen Daten in Bezug auf Quantität, Qualität und Komplexität exponentiell zunehmen. In dieser Arbeit haben wir in drei Projekten, die auf die Charakterisierung von funktionellen Eigenschaften von Membrantransportsystemen sowie deren Wechselwirkungen mit Substraten und Nicht-Substraten abzielen, Bioinformatik-Werkzeuge/-Techniken entwickelt, umgesetzt und angewendet. Membrantransporter sind eine sehr wichtige Klasse von integralen Transmembranproteinen, die für den Materialaustausch zwischen Zellen und deren Umgebungen verantwortlich sind. Aufgrund der starken Beziehung mit verschiedenen Krankheiten und abnormen medizinischen Bedingungen wurde und wird die Wechselwirkung von Transportern mit kleinen Arzneimittelmolekülen intensiv untersucht. Im ersten Projekt haben wir eine neuartige Methode für die MdfA-Substratklassifizierung entwickelt. MdfA ist ein Multidrug-Membrantransporter von E. coli, der für die Erkennung und den Transport eines breiten Spektrums von Substraten mit nicht verwandten Eigenschaften verantwortlich ist. Im Gegensatz zu anderen herkömmlichen Verfahren, die allgemeine Merkmale wie aus den sequenzen abgeleitete Informationen, molekulare Deskriptoren usw. verwenden, umfasst das neue Verfahren Protein- Ligand-Struktur-Wechselwirkungen und potentielle Energieinformationen, die aus molekulardynamischen Simulationen abgeleitet sind. Allerdings stieß das Verfahren immer noch auf Schwierigkeiten mit dem strukturellen Ähnlichkeitsproblem zwischen Substraten und Nichtsubstraten. Die neue Methode erreichte eine zufriedenstellende Genauigkeit mit 73,12% Klassifizierungsgenauigkeit. Es ist die erste Methode, die Protein-Ligand-Wechselwirkungen bei einem Klassifizierungsproblem für Membrantransport berücksichtigt. Im nÄchsten Projekt analysierten wir Proteomikdaten aus Sec61α und TRAP-Stummschaltungsexperimenten, um TRAP-Substrate zu identifizieren und zu charakterisieren. TRAP ist eine assistierende Komponente des Translocon-Komplexes, der für die Protein-Translokation verantwortlich ist. Wir identifizierten erfolgreich einen Satz von TRAP-abhÄngigen Proteinen aus Massenspektrometrie-Proteomik-Daten. Darüber hinaus zeigte unsere Analyse, dass die Signalpeptide von TRAP-Substraten eine geringe Hydrophobie-Tendenz sowie einen signifikant erhöhten Glycin- und Prolin-Gehalt aufwiesen. Wir schlugen vor, dass TRAP dafür verantwortlich sein VII kann, diejenigen Proteine bei der Migration durch den Sec61α-Kanal zu unterstützen, die Signalpeptide mit hohem Glycin-Prolin-Gehalt und geringer HydrophobizitÄt haben. Im letzten Projekt haben wir die molekulare Docking-Technik angewendet, um die Bindungsmodi von mehreren Eeyarestatin-Verbindungen (ES1, ES24, ES35 und ES47) mit einem Homologiemodell von humanem Sec61α Protein zu untersuchen. Der Sec61α-Kanal ist nicht nur für die Proteintranslokation verantwortlich, sondern fördert auch Ca2+ Leckage. Die Docking-Ergebnisse ergaben, dass sich die energetisch günstigste Bindungsposition von ES1 und ES24 zwischen den H2- und H7- Helices befindet, die die \Türen" des lateralen Tores sind. Daher ist es wahrscheinlich, dass sie die Tor-Funktion behindern können und nach der Bindung den Kanal offen halten. Daher haben wir postuliert, dass ES1 und ES24 die potentiellen \Gate Blocker" sein können, die Ca2+ Leckage durch Sec61α fördern. Diese Ergebnisse stimmen mit den Ergebnissen der Calcium-Imaging-Experimente überein, die von unseren Kollegen durchgeführt wurden. In dieser Arbeit haben wir verschiedene Rechentechniken eingesetzt, um neue mechanistische Einblicke in Transmembran-Transporter zu gewinnen und wichtige Informationen aus der Analyse von Proteomik-Daten zu erhalten. Wir hoffen, dass unsere Arbeit nützliche mikroskopische Details und mögliche Mechanismen für die experimentellen Biologen, die an transmembranen Proteinen arbeiten, zur Verfügung stellt.SFB 1027, GRK127

Interaction Of Isoniazid With Mycobacterium Tuberculosis Enoyl-Acyl Carrier Protein Reductase (INHA) : From Molecular Perspectives [RA644.T7 C548 2008 f rb].

Masalah kerintangan ubat dan peningkatan kes-kes tibi yang berterusan telah menggalakkan penyelidikan pembangunan ubat baru dan meningkatkan pemahaman mekanisme kerintangan ubat. The problem of tuberculosis (TB) drug resistance and the continuing rise in the disease incidence has prompted the research on new drug development as well as on increasing the understanding of the mechanisms of drug resistance

Structural effects of clinically observed mutations in JAK2 exons 13-15: comparison with V617F and exon 12 mutations

<p>Abstract</p> <p>Background</p> <p>The functional relevance of many of the recently detected JAK2 mutations, except V617F and exon 12 mutants, in patients with chronic myeloproliferative neoplasia (MPN) has been significantly overlooked. To explore atomic-level explanations of the possible mutational effects from those overlooked mutants, we performed a set of molecular dynamics simulations on clinically observed mutants, including newly discovered mutations (K539L, R564L, L579F, H587N, S591L, H606Q, V617I, V617F, C618R, L624P, whole exon 14-deletion) and control mutants (V617C, V617Y, K603Q/N667K).</p> <p>Results</p> <p>Simulation results are consistent with all currently available clinical/experimental evidence. The simulation-derived putative interface, not possibly obtained from static models, between the kinase (JH1) and pseudokinase (JH2) domains of JAK2 provides a platform able to explain the mutational effect for all mutants, including presumably benign control mutants, at the atomic level.</p> <p>Conclusion</p> <p>The results and analysis provide structural bases for mutational mechanisms of JAK2, may advance the understanding of JAK2 auto-regulation, and have the potential to lead to therapeutic approaches. Together with recent mutation profiling results demonstrating the breadth of clinically observed JAK2 mutations, our findings suggest that molecular testing/diagnostics of JAK2 should extend beyond V617F and exon 12 mutations, and perhaps should encompass most of the pseudo-kinase domain-coding region.</p

The role of electrostatics in the mechanism of ATP/ADP carrier function: an in silico study

Tese de mestrado em Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2020As forças electrostáticas têm um papel fulcral numa extensa panóplia de processos biomoleculares desde expressão de genes, resposta imune, condução de moléculas até centros catalíticos, acoplamento de subunidades, entre outros. A grande ubiquidade destas prende-se com a sua habilidade de actuarem a longas distâncias. Assim sendo, a capacidade de descrever as forças electrostáticas associadas aos diversos processos é uma ferramenta extremamente útil, porém, infelizmente, o seu estudo com detalhe atomístico é bastante difícil de realizar através de métodos experimentais. Desta forma os métodos computacionais têm vindo a ganhar relevância tanto pelo grande aumento de poder computacional nas últimas décadas, como também pela sua capacidade de colmatar esta lacuna a nível experimental. Com isto em mente, o presente trabalho pretende demonstrar um protocolo computacional efectivo no estudo das propriedades electrostáticas de sistemas moleculares de grandes dimensões e complexidade. O objecto de estudo escolhido para a validação deste protocolo foi a proteína transportadora de ATP/ADP (AAC), responsável por importar o ADP para a matriz mitocondrial e exportar ATP da matriz para o espaço inter-membranar do mitocôndrio. Sendo capaz de atrair e transportar moléculas altamente negativas aponta imediatamente para a presença de uma superfície electrostática complexa e essencial para que o processo ocorra eficientemente. A somar às características mencionadas anteriormente, o facto deste transportador estar bem caracterizado e estudado torna-o um bom candidato a usar na demonstração de um protocolo de estudo da electrostática associada a processos biomoleculares. Para concretizar este estudo três técnicas computacionais foram utilizadas funcionando em complementaridade. Primeiramente simulações longas de dinâmica molecular foram realizadas, após a inserção do transportador de ATP/ADP em membranas de POPC. Com estas torna-se possível avaliar a estabilidade do nosso sistema proteína-membrana, verificando a viabilidade das condições escolhidas. Efectivamente foi observado que o uso do método de campo de força generalizado para o tratamento da electrostática a longa distância introduziu instabilidades estruturais nas simulações. Assim, foi necessário readaptar o nosso protocolo aplicando o método de somas de Ewald com malha de partículas para o tratamento da electrostática a longa distância. Procedendo a esta alteração no tratamento das nossas simulações torna-se possível equilibrar o nosso sistema de modo a ser empregue na técnica de Dinâmica Molecular a pH Constante (CpHMD). Esta acrescenta um nível de detalhe à descrição do sistema ao permitir a ocorrência de eventos de (des)protonação numa lista definida de moléculas e/ou resíduos de aminoácido. Realizando simulações de CpHMD em ambos os estados, C e M, do AAC em forma apo e a quatro valores de pH, 4, 5, 6 e 7, torna-se possível obter curvas de titulação e valores de pKa para diversos aminoácidos presentes no transportador de membrana. Analisando a presença de desvios nos pKa detectados nas nossas simulações de CpHMD torna-se possível a discussão acerca do seu ambiente circundante e de possíveis interacções electrostáticas entre resíduos da proteína. Destas simulações fomos capazes de identificar um grande desvio no pKa da lisina 22, trazendo o seu valor de 10.4, quando solvatado, para ∼ 8. Este desvio fora já reportado em publicações anteriores e, à semelhança do nosso trabalho, demonstra-se relevante para a actividade do transportador, uma vez que a forma protonada deste resíduo é essencial à actividade. Das nossas análises foi também possível identificar três resíduos ácidos na proteína cujo pKa se encontrava desviado para valores inferiores no estado C do AAC. Este desvio indica-nos a presença de interacções electrostáticas que estabilizam o estado carregado dos resíduos glutamato 29, aspartato 134 e aspartato 231. Efectivamente estas interacções consistem na formação de pontes salinas que ajudam no fecho da cavidade do AAC no estado C. Com estes resultados é possível demonstrar a utilidade do estudo de uma proteína através da técnica de CpHMD, sendo possível identificar resíduos chave para a actividade do transportador e interações cruciais para o bom funcionamento da proteína. Finalmente, pretendemos não só estudar a forma apo do AAC mas também o processo inteiro de transporte dos substratos através da proteína transmembranar com o intuito de obter uma descrição energética do processo e em simultâneo captar informações acerca das forças electrostáticas envolvidas neste. No entanto, as simulações tanto de MD como de CpHMD apenas são viáveis em pequenas escalas temporais, à volta de poucos microssegundos, já o tempo total do processo de transporte poderá chegar aos milissegundos. De forma a contornar este problema, será usada uma técnica de amostragem aumentada, Umbrella Sampling (US). Nesta, o processo de transporte é dividido em várias janelas segundo uma coordenada de reação e um potencial de enviesamento é aplicado ao substrato para o manter na posição definida. Desta forma, somos capazes de extrair a descrição energética do processo e os perfis de protonação não só do substrato como também dos resíduos do transportador de ATP/ADP. Conjugando os resultados dos dois tipos de transporte, importação de substrato para a matriz e exportação de substrato da matriz, para cada substrato, ATP e ADP, é possível comparar a totalidade de condições de transporte e esclarecer detalhes acerca do mecanismo executado pela proteína. Com as nossas simulações foi possível concluir que o mecanismo de importação dos substratos beneficia de diversos detalhes que não se encontram presentes no processo de exportação. Em primeira instância, através dos resultados obtidos da força potencial média (PMF) e da protonação de ambos os substratos, identificou-se a presença de um maior potencial electrostático positivo na cavidade do estado C, responsável pela captação de substrato no processo de importação. Este potencial conseguirá atrair os substratos do citoplasma para a cavidade a maiores distâncias do que o estado M. Aquando da comparação dos perfis energéticos de importação, foi observada a preferência energética da importação de ADP pelo seu perfil energético inferior relativamente ao do ATP. Já no caso da exportação, ambos apresentam perfis bastante semelhantes, não havendo um favorecimento do transporte de um substrato em particular. Por fim, os dois processos de transporte distinguem-se entre si também ao nível das redes de pontes salinas que executam o fecho da cavidade do estado C e M. Considerando o processo de importação, onde o estado C é responsável pela atração das moléculas, verificou-se a existência da rede de pontes salinas directamente no fundo da cavidade. Esta é constituída pelos resíduos ácidos mencionados nos resultados do CpHMD, Glu29, Asp134 e Asp231. Foi verificado que, ao ocorrer a aproximação do substrato ao fundo da cavidade do estado C, estes ácidos têm tendência a protonarem, o que leva, por consequência, ao enfraquecimento das pontes salinas e a uma mais rápida transição conformacional do estado C para o estado M. Em contrapartida, no processo de exportação, onde o estado M é responsável pela atracção dos substratos, a localização das pontes salinas encontra-se na extremidade citoplasmática da proteína, sendo assim insensível à presença do substrato no fundo da cavidade. Estas características distintas entre o estado C e M, relevantes para o processo de importação e exportação respectivamente, desenham um panorama que evidencia maior dificuldade na importação de substratos. In vivo, este processo é realizado ao atrair o ADP de um meio vasto e escasso em substrato junto da abertura do AAC para o interior da cavidade do estado C. Desta forma, a existência de um maior potencial positivo na cavidade, capaz de captar efectivamente o substrato, a presença de uma armadilha energética que previne a libertação natural do ADP de volta para o meio envolvente e uma mais rápida transição conformacional devido ao enfraquecimento das pontes salinas, aquando da aproximação do substrato, são características essenciais para que o processo de importação se dê com a eficiência e rapidez necessárias. Em contraste, no processo de exportação, o estado M atrai o ATP da matriz mitocôndrial, cujo volume é mais reduzido e onde existe uma grande abundância de substrato. Desta forma este estado não é submetido à pressão do ambiente para evoluir no sentido de aumentar a rapidez e eficiência do transporte, reflectindo-se na ausência das características observadas no estado C. Concluindo este trabalho, foi-nos possível demonstrar como o uso de técnicas computacionais permite um estudo detalhado e intensivo de um sistema de grandes proporções e complexidade, fornecendo informações cruciais acerca das forças electrostáticas e obtendo perfis energéticos que auxiliam na construção do mecanismo de acção da proteína. No presente caso, o protocolo foi usado numa proteína bem estudada e muito do seu mecanismo já fora elucidado. Porém, a aplicação deste mesmo protocolo numa proteína menos estudada poderá vir a revelar importantes detalhes sobre o seu mecanismo e levar à identificação de detalhes estruturais essenciais à actividade da proteína.To accurately describe the electrostatic interactions with atomistic detail is an exceedingly useful tool, since they play a major role in all biomolecular processes. In the present work, we introduced a new computational protocol based on Constant pH Molecular Dynamics (CpHMD) coupled to an Umbrella Sampling scheme (US-CpHMD). Using these techniques, we modeled the conformational changes of ATP/ADP carrier (AAC) coupled to the most relevant protonation events and even extracted the equilibrium energetics involved in the transport of ATP and ADP across the inner mitochondrial membrane. The transport activity of this protein is deeply connected with electrostatic interactions involved in the binding of the highly negative charged substrates. CpHMD simulations were performed on pre-equilibrated apo-AAC:POPC systems, allowing the extraction of titration curves and pKa values of several AAC residues. We were able to reproduce the reported pKa shift of Lys22 from the water soluble pKa value (10.4) to ∼ 8, which makes this residue the responsible for the sensitivity of this protein to basic pH values, modulating the activity of AAC. Three acid aminoacids, Glu29, Asp134 and Asp231, also showed shifted pKa values which revealed their role in establishing electrostatic interactions in the bottom of AAC cavity, forming the matrix salt-bridge network responsible for closing the C-state cavity. Taking a step further, US-CpHMD was used to mimic the transport of substrates while capturing conformational and electrostatic effects. Clear differences between the import and export process were detected in our analysis which are connected to the distinct evolutionary pressures of each transport. The import has developed a larger positive electrostatic potential in the cavity and a clear selectivity towards ADP in order to efficiently capture this molecule. In addition, we observed a substrate-induced shift in the protonation of the acid residues present on the matrix salt-bridge network. This protonation triggers the conformational transition from C- to M-state by weakening the matrix salt-bridges, leading to an acceleration of the import. These characteristics were of high importance for a quick and efficient import process, which is crucial due to the low abundance of the ADP near the C-state cavity. In contrast, from these features only a slightly weaker positive potential was present on the export process. The absence of the other traits is tied with the high abundance of ATP molecules near the M-state cavity and its almost unneeded selectivity over ADP, which is caused by a significantly lower magnitude of evolutionary pressure exerted in this process. Hence the M-state did not develop the same traits as the C-state. The entirety of results showed the success of employing this computational protocol in the analysis of mechanistic details at the atomic level, enabling the extraction of key electrostatic interactions and energetic profiles of the biomolecular process. The application of this protocol to less studied proteins and processes may prove highly advantageous, possibly aiding in the elucidation of their mechanism and key electrostatic details

MOLECULAR DYNAMICS ANALYSIS OF THE BEHAVIOUR OF PROLYL OLIGOPEPTIDASE (POP) IN THE PRESENCE OF Z-PRO-PROLINAL INHIBITOR

Prolyl Oligopeptidase (POP), a member of the prolyl endopeptidase family, has a role in several neurological disorders. Its primary function is to cleave an oligopeptidase, including neuroactive peptides. On the other hand POP with a Z-Pro-prolinal (ZPP) inhibitor may revert memory loss from neurological disorders, amnesic agents and aging. Here, the crystal structure of POP protein with ZPP inhibitor (Protein Data Bank PDB) and without ZPP inhibitor is studied using classical molecular dynamics simulations and the POP-ZPP complex behaviour is compared with pure POP. The basic analysis of the structures, included measuring radius of gyration and root mean square deviation which proved that POP structure with non-bonded ZPP and without ZPP are stable and maintain their structure over the entire simulation time. Moreover, principal component analysis (PCA) is used to analyze the motions of the structures by extracting the normal modes of motions in POP with and without presence of ZPP inhibitor

Predicting locations of cryptic pockets from single protein structures using the PocketMiner graph neural network

Cryptic pockets expand the scope of drug discovery by enabling targeting of proteins currently considered undruggable because they lack pockets in their ground state structures. However, identifying cryptic pockets is labor-intensive and slow. The ability to accurately and rapidly predict if and where cryptic pockets are likely to form from a structure would greatly accelerate the search for druggable pockets. Here, we present PocketMiner, a graph neural network trained to predict where pockets are likely to open in molecular dynamics simulations. Applying PocketMiner to single structures from a newly curated dataset of 39 experimentally confirmed cryptic pockets demonstrates that it accurately identifies cryptic pockets (ROC-AUC: 0.87) \u3e1,000-fold faster than existing methods. We apply PocketMiner across the human proteome and show that predicted pockets open in simulations, suggesting that over half of proteins thought to lack pockets based on available structures likely contain cryptic pockets, vastly expanding the potentially druggable proteome