RESUMO
Therapeutic interventions are designed to perturb the function of a biological system. However, there are many types of proteins that cannot be targeted with conventional small molecule drugs. Accordingly, many identified gene-regulatory drivers and downstream effectors are currently undruggable. Drivers and effectors are often connected by druggable signaling and regulatory intermediates. Methods to identify druggable intermediates therefore have general value in expanding the set of targets available for hypothesis-driven validation. Here we identify and prioritize potential druggable intermediates by developing a network perturbation theory, termed NetPert, for response functions of biological networks. Dynamics are defined by a network structure in which vertices represent genes and proteins, and edges represent gene-regulatory interactions and protein-protein interactions. Perturbation theory for network dynamics prioritizes targets that interfere with signaling from driver to response genes. Applications to organoid models for metastatic breast cancer demonstrate the ability of this mathematical framework to identify and prioritize druggable intermediates. While the short-time limit of the perturbation theory resembles betweenness centrality, NetPert is superior in generating target rankings that correlate with previous wet-lab assays and are more robust to incomplete or noisy network data. NetPert also performs better than a related graph diffusion approach. Wet-lab assays demonstrate that drugs for targets identified by NetPert, including targets that are not themselves differentially expressed, are active in suppressing additional metastatic phenotypes.
Assuntos
Neoplasias da Mama , Biologia Computacional , Redes Reguladoras de Genes , Humanos , Redes Reguladoras de Genes/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Transdução de Sinais/efeitos dos fármacos , Modelos Biológicos , Antineoplásicos/farmacologia , FemininoRESUMO
A lesson utilizing a coarse-grained (CG) Go-like model has been implemented into the CHARMM INterface and Graphics (CHARMMing) web portal (www.charmming.org) to the Chemistry at HARvard Macromolecular Mechanics (CHARMM) molecular simulation package. While widely used to model various biophysical processes, such as protein folding and aggregation, CG models can also serve as an educational tool because they can provide qualitative descriptions of complex biophysical phenomena for a relatively cheap computational cost. As a proof of concept, this lesson demonstrates the construction of a CG model of a small globular protein, its simulation via Langevin dynamics, and the analysis of the resulting data. This lesson makes connections between modern molecular simulation techniques and topics commonly presented in an advanced undergraduate lecture on physical chemistry. It culminates in a straightforward analysis of a short dynamics trajectory of a small fast folding globular protein; we briefly describe the thermodynamic properties that can be calculated from this analysis. The assumptions inherent in the model and the data analysis are laid out in a clear, concise manner, and the techniques used are consistent with those employed by specialists in the field of CG modeling. One of the major tasks in building the Go-like model is determining the relative strength of the nonbonded interactions between coarse-grained sites. New functionality has been added to CHARMMing to facilitate this process. The implementation of these features into CHARMMing helps automate many of the tedious aspects of constructing a CG Go model. The CG model builder and its accompanying lesson should be a valuable tool to chemistry students, teachers, and modelers in the field.
Assuntos
Biologia Computacional/educação , Biologia Computacional/métodos , Internet , Dobramento de Proteína , Software , Simulação de Dinâmica Molecular , Proteínas/química , Proteínas/metabolismo , Interface Usuário-ComputadorRESUMO
The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Difusão , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , HidrodinâmicaRESUMO
The bilayer bending modulus (Kc) is one of the most important physical constants characterizing lipid membranes, but precisely measuring it is a challenge, both experimentally and computationally. Experimental measurements on chemically identical bilayers often differ depending upon the techniques employed, and robust simulation results have previously been limited to coarse-grained models (at varying levels of resolution). This Communication demonstrates the extraction of Kc from fully atomistic molecular dynamics simulations for three different single-component lipid bilayers (DPPC, DOPC, and DOPE). The results agree quantitatively with experiments that measure thermal shape fluctuations in giant unilamellar vesicles. Lipid tilt, twist, and compression moduli are also reported.
Assuntos
Bicamadas Lipídicas/química , Simulação de Dinâmica MolecularRESUMO
Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared with matched noninvasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified 3 distinct transcriptomic groups in invasive organoids, 2 of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGF-ß1, IL-6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Transcriptoma , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/metabolismo , Organoides/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Microambiente Tumoral/genéticaRESUMO
Hepatocellular carcinoma (HCC) is the sixth most common and the fourth most deadly cancer worldwide. The development cost of new therapeutics is a major limitation in patient outcomes. Importantly, there is a paucity of preclinical HCC models in which to test new small molecules. Herein, we implemented potentially novel patient-derived organoid (PDO) and patient-derived xenografts (PDX) strategies for high-throughput drug screening. Omacetaxine, an FDA-approved drug for chronic myelogenous leukemia (CML), was found to be a top effective small molecule in HCC PDOs. Next, omacetaxine was tested against a larger cohort of 40 human HCC PDOs. Serial dilution experiments demonstrated that omacetaxine is effective at low (nanomolar) concentrations. Mechanistic studies established that omacetaxine inhibits global protein synthesis, with a disproportionate effect on short-half-life proteins. High-throughput expression screening identified molecular targets for omacetaxine, including key oncogenes, such as PLK1. In conclusion, by using an innovative strategy, we report - for the first time to our knowledge - the effectiveness of omacetaxine in HCC. In addition, we elucidate key mechanisms of omacetaxine action. Finally, we provide a proof-of-principle basis for future studies applying drug screening PDOs sequenced with candidate validation in PDX models. Clinical trials could be considered to evaluate omacetaxine in patients with HCC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular , Mepesuccinato de Omacetaxina/farmacologia , Neoplasias Hepáticas , Adulto , Idoso , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Organoides/efeitos dos fármacos , Organoides/patologia , Inibidores da Síntese de Proteínas/farmacologia , Adulto JovemRESUMO
Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. To test this hypothesis, we use single-molecule fluorescence microscopy to probe the effects of lipid binding stoichiometry on the diffusion constants of engineered proteins containing one to three pleckstrin homology domains coupled by flexible linkers. Within error, the lateral diffusion constants of these engineered constructs are inversely proportional to the number of tightly bound phosphatidylinositol-(3,4,5)-trisphosphate lipids. The same trend is observed in coarse-grained molecular dynamics simulations and hydrodynamic bead calculations of lipid multimers connected by model tethers. Overall, single molecule diffusion measurements are found to provide molecular information about protein-lipid interactions. Moreover, the experimental and computational results independently indicate that the frictional contributions of multiple, coupled but well-separated lipids are additive, analogous to the free-draining limit for isotropic fluids--an insight with significant implications for theoretical description of bilayer lipid dynamics.
Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Fenômenos Biofísicos , Difusão Facilitada , Hidrodinâmica , Proteínas de Membrana/genética , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Ligação Proteica , Engenharia de Proteínas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The periodic Saffman-Delbrück (PSD) model, an extension of the Saffman-Delbrück model developed to describe the effects of periodic boundary conditions on the diffusion constants of lipids and proteins obtained from simulation, is tested using the coarse-grained Martini and all-atom CHARMM36 (C36) force fields. Simulations of pure Martini dipalmitoylphosphatidylcholine (DPPC) bilayers and those with one embedded gramicidin A (gA) dimer or one gA monomer with sizes ranging from 512 to 2048 lipids support the PSD model. Underestimates of D∞ (the value of the diffusion constant for an infinite system) from the 512-lipid system are 35% for DPPC, 45% for the gA monomer, and 70% for the gA dimer. Simulations of all-atom DPPC and dioleoylphosphatidylcholine (DOPC) bilayers yield diffusion constants not far from experiment. However, the PSD model predicts that diffusion constants at the sizes of the simulation should underestimate experiment by approximately a factor of 3 for DPPC and 2 for DOPC. This likely implies a deficiency in the C36 force field. A Bayesian method for extrapolating diffusion constants of lipids and proteins in membranes obtained from simulation to infinite system size is provided.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Gramicidina/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Difusão , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Developing methods to incorporate protein flexibility into structure-based drug design is an important challenge. Our approach uses multiple protein structures (MPS) to create a receptor-based pharmacophore model of the desired target. We have previously demonstrated the success of the method by applying it to human immunodeficiency virus-1 protease (HIV-1p). Our models, based on an apo structure, discriminated known HIV-1p inhibitors from druglike inactive compounds and also accurately identified bound conformations of known inhibitors. Here, we test the method by applying it to all three unbound crystal structures of HIV-1p. We have also improved our method with denser probe mapping of the binding site and refined our selection criteria for pharmacophore elements. Our improved protocol has led to the development of a consistent 8-site pharmacophore model for HIV-1p, which is independent of starting structure, and a robust MPS pharmacophore method that is more amenable to automation.
Assuntos
Inibidores da Protease de HIV/química , Protease de HIV/química , HIV-1/enzimologia , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Ligantes , Conformação ProteicaRESUMO
Binding MOAD (Mother of All Databases) is the largest collection of high-quality, protein-ligand complexes available from the Protein Data Bank. At this time, Binding MOAD contains 5331 protein-ligand complexes comprised of 1780 unique protein families and 2630 unique ligands. We have searched the crystallography papers for all 5000+ structures and compiled binding data for 1375 (26%) of the protein-ligand complexes. The binding-affinity data ranges 13 orders of magnitude. This is the largest collection of binding data reported to date in the literature. We have also addressed the issue of redundancy in the data. To create a nonredundant dataset, one protein from each of the 1780 protein families was chosen as a representative. Representatives were chosen by tightest binding, best resolution, etc. For the 1780 "best" complexes that comprise the nonredundant version of Binding MOAD, 475 (27%) have binding data. This significant collection of protein-ligand complexes will be very useful in elucidating the biophysical patterns of molecular recognition and enzymatic regulation. The complexes with binding-affinity data will help in the development of improved scoring functions and structure-based drug discovery techniques. The dataset can be accessed at http://www.BindingMOAD.org.
Assuntos
Biofísica/métodos , Biologia Computacional/métodos , Bases de Dados de Proteínas , Proteômica/métodos , Análise por Conglomerados , Cristalografia por Raios X , Bases de Dados Bibliográficas , Internet , Cinética , Ligantes , Modelos Moleculares , Ligação ProteicaRESUMO
Use of solvent mapping, based on multiple-copy minimization (MCM) techniques, is common in structure-based drug discovery. The minima of small-molecule probes define locations for complementary interactions within a binding pocket. Here, we present improved methods for MCM. In particular, a Jarvis-Patrick (JP) method is outlined for grouping the final locations of minimized probes into physical clusters. This algorithm has been tested through a study of protein-protein interfaces, showing the process to be robust, deterministic, and fast in the mapping of protein "hot spots." Improvements in the initial placement of probe molecules are also described. A final application to HIV-1 protease shows how our automated technique can be used to partition data too complicated to analyze by hand. These new automated methods may be easily and quickly extended to other protein systems, and our clustering methodology may be readily incorporated into other clustering packages.
Assuntos
Algoritmos , Desenho de Fármacos , Modelos Moleculares , Proteínas/química , Solventes/química , Benzeno/química , Domínio Catalítico , Protease de HIV/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Escherichia coli dihydrofolate reductase (DHFR) is a long-standing target for enzyme studies. The influence of protein motion on its catalytic cycle is significant, and the conformation of the M20 loop is of particular interest. We present receptor-based pharmacophore models-an equivalent of solvent-mapping of binding hotspots-based on ensembles of protein conformations from molecular dynamics simulations of DHFR.NADPH in both the closed and open conformation of the M20 loop. The optimal models identify DHFR inhibitors over druglike non-inhibitors; furthermore, high-affinity inhibitors of E. coli DHFR are preferentially identified over general DHFR inhibitors. As expected, models resulting from simulations with DHFR in the productive conformation with a closed M20 loop have better performance than those from the open-loop simulations. Model performance improves with increased dynamic sampling, indicating that including a greater degree of protein flexibility can enhance the quest for potent inhibitors. This was compared to the limited conformational sampling seen in crystal structures, which were suboptimal for this application.
Assuntos
Desenho de Fármacos , Escherichia coli/enzimologia , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Cristalografia por Raios X , Bases de Dados de Proteínas , Ativação Enzimática/efeitos dos fármacos , Ligantes , Modelos Moleculares , Conformação MolecularRESUMO
In structure-based drug discovery, researchers would like to identify all possible scaffolds for a given target. However, techniques that push the boundaries of chemical space could lead to many false positives or inhibitors that lack specificity for the target. Is it possible to broadly identify the appropriate chemical space for the inhibitors and yet maintain target specificity? To address this question, we have turned to dihydrofolate reductase (DHFR), a well-studied metabolic enzyme of pharmacological relevance. We have extended our multiple protein structure (MPS) method for receptor-based pharmacophore models to use multiple X-ray crystallographic structures. Models were created for DHFR from human and Pneumocystis carinii. These models incorporate a fair degree of protein flexibility and are highly selective for known DHFR inhibitors over drug-like non-inhibitors. Despite sharing a highly conserved active site, the pharmacophore models reflect subtle differences between the human and P. carinii forms, which identify species-specific, high-affinity inhibitors. We also use structures of DHFR from Candida albicans as a counter example. The available crystal structures show little flexibility, and the resulting models give poorer performance in identifying species-specific inhibitors. Therapeutic success for this system may depend on achieving species specificity between the related human host and these key fungal targets. The MPS technique is a promising advance for structure-based drug discovery for DHFR and other proteins of biomedical interest.