Inhibition of protein interactions: co-crystalized protein–protein interfaces are nearly as good as holo proteins in rigid-body ligand docking

Modulating protein interaction pathways may lead to the cure of many diseases. Known protein–protein inhibitors bind to large pockets on the protein–protein interface. Such large pockets are detected also in the protein–protein complexes without known inhibitors, making such complexes potentially druggable. The inhibitor-binding site is primary defined by the side chains that form the largest pocket in the protein-bound conformation. Low-resolution ligand docking shows that the success rate for the protein-bound conformation is close to the one for the ligand-bound conformation, and significantly higher than for the apo conformation. The conformational change on the protein interface upon binding to the other protein results in a pocket employed by the ligand when it binds to that interface. This proof-of-concept study suggests that rather than using computational pocket-opening procedures, one can opt for an experimentally determined structure of the target co-crystallized protein–protein complex as a starting point for drug design

Drug repurposing to target Ebola virus replication and virulence using structural systems pharmacology

Background: The recent outbreak of Ebola has been cited as the largest in history. Despite this global health crisis, few drugs are available to efficiently treat Ebola infections. Drug repurposing provides a potentially efficient solution to accelerating the development of therapeutic approaches in response to Ebola outbreak. To identify such candidates, we use an integrated structural systems pharmacology pipeline which combines proteome-scale ligand binding site comparison, protein-ligand docking, and Molecular Dynamics (MD) simulation. Results: One thousand seven hundred and sixty-six FDA-approved drugs and 259 experimental drugs were screened to identify those with the potential to inhibit the replication and virulence of Ebola, and to determine the binding modes with their respective targets. Initial screening has identified a number of promising hits. Notably, Indinavir; an HIV protease inhibitor, may be effective in reducing the virulence of Ebola. Additionally, an antifungal (Sinefungin) and several anti-viral drugs (e.g. Maraviroc, Abacavir, Telbivudine, and Cidofovir) may inhibit Ebola RNA-directed RNA polymerase through targeting the MTase domain. Conclusions: Identification of safe drug candidates is a crucial first step toward the determination of timely and effective therapeutic approaches to address and mitigate the impact of the Ebola global crisis and future outbreaks of pathogenic diseases. Further in vitro and in vivo testing to evaluate the anti-Ebola activity of these drugs is warranted

Improving the accuracy and efficiency of docking methods

Computational methods for predicting macromolecular complexes are useful tools for studying biological systems. They are used in areas such as drug design and for studying protein-protein interactions. While considerable progress has been made in this field over the decades, enhancing the speed and accuracy of these computational methods remains an important challenge. This work describes two different enhancements to the accuracy of ClusPro, a method for performing protein-protein docking, as well as an enhancement to the efficiency of global rigid body docking. SAXS is a high throughput technique collected for molecules in solution, and the data provides information about the shape and size of molecules. ClusPro was enhanced with the ability to SAXS data collected for protein complexes to guide docking by selecting conformations by how well they match the experimental data, which improved docking accuracy when such data is available. Various other experimental techniques, such as NMR, FRET, or chemical cross linking can provide information about protein-protein interfaces, and such information can be used to generate distance-based restraints between pairs of residues across the interface. A second enhancement to ClusPro enables the use of such distance restraints to improve docking accuracy. Finally, an enhancement to the efficiency of FFT based global docking programs was developed. This enhancement allows for the efficient search of multiple sidechain conformations, and this improved program was applied to the flexible computational solvent mapping program FTFlex.2018-07-09T00:00:00

Computational modeling of protein-protein and protein-peptide interactions

Protein-protein and protein-peptide interactions play a central role in various aspects of the structural and functional organization of the cell. While the most complete structural characterization is provided by X-ray crystallography, many biological interactions occur in complexes that will not be amenable to direct experimental analysis. Therefore, it is important to develop computational docking methods that start from the structures of component proteins and predict the structure of their complexes, preferably with accuracy close to that provided by X-ray crystallography. This thesis details three applications of computational protein modeling, including the study of antibody maturation mechanisms, and the development of protocols for peptide-protein interaction prediction and template-based modeling of protein complexes. The first project, a comparative analysis of docking an antigen structure to antibodies across a lineage, reveals insights into antibody maturation mechanisms. A linear relationship between near-native docking results and changes in binding free energy is established, and used to investigate changes in binding affinity following mutation across two antibody-antigen systems: influenza and anthrax. The second project demonstrates that a motif-based search of available protein crystal structures is sufficient to adequately represent the conformational space sampled by a flexible peptide, compared to that of a rigid globular protein. This observation forms the basis for a global peptide-protein docking protocol that has since been implemented into the Structural Bioinformatics Laboratory’s docking web server, ClusPro. Finally, as structure availability remains a roadblock to many studies, researchers turn to homology modeling, in which the desired protein sequence is modeled onto a related structure. This is particularly challenging when the target is a protein complex, further restricting template availability. To address this problem, the third project details the development of a new template-based modeling protocol to be integrated into the ClusPro server. The implementation of a novel template-based search enables users to model both homomeric and heteromeric complexes, greatly expanding ClusPro server functionality.2020-08-30T00:00:00

Activation and Inhibition of Biological Function through Design of Novel Protein-Ligand Interactions

Virtually every process within a cell involves a protein. They serve as cellular workhorses carrying out functions such as catalysis of essential metabolites, to regulating which genes get turned on or off, to forming the structural scaffolding to retain rigidity of a cell. Proteins form the link between the genetic information encoded in DNA to the observable phenotype of an organism. The way proteins communicate is by direct physical contact with another molecule that alters its shape and dynamics to carry out a particular function. For example, G protein-coupled receptors are membrane imbedded proteins that bind to a small molecule or peptide in the extracellular environment and translate the binding event into an internal signal to regulate processes such as heart rate and even mood. The ability to selectively modulate such fundamental systems offers huge potential with broad applications from the ability to interrogate unknown cellular mechanisms to developing therapeutics when these interactions become aberrant. The scope of this dissertation encompasses determining what properties dictate protein-ligand interactions and the application of these principles to the design of new ones. In particular, chapter 1 covers the design of a molecular switch that is turned on by small molecules. I follow this up in chapter 2 by investigating how to turn off protein function with small molecules in aberrant disease states. In chapter 3 we expand from the world of small molecule ligands to design a protein to turn off function of a protein involved in bacterial pathogenesis

Evidence of Conformational Selection Driving the Formation of Ligand Binding Sites in Protein-Protein Interfaces

<div><p>Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved.</p></div

Role of a highly conserved region of the NF-kappaB essential modulator in its scaffolding function

Scaffold proteins facilitate many aspects of intracellular signaling. These proteins can regulate two or more proteins in the same pathway, or coordinate signaling from multiple pathways. Scaffold proteins are therefore key control points for the flux of signaling and play essential roles in biological systems. There are four possible mechanisms by which scaffold proteins achieve activation and propagate signaling: 1) rigid protein binding between two or more proteins to co-localize binding partners, 2) ligand-induced activation such as may result from a conformational change, 3) disorder-to-order transition where the scaffold protein folds as a result of a protein-protein interaction, and 4) dynamic processes such as phosphorylation. The scaffold protein NF-κB essential modulator (NEMO) functions via ligand-induced activation and serves as the key control point for canonical NF-κB signaling. The work described in this thesis investigates the role of a previously uncharacterized domain within NEMO that is required for function, which we term the Intervening Domain (IVD). Bioinformatic analysis reveals a high level of sequence conservation across species within this domain. Conformational changes following ligand binding are observed for NEMO and these changes require conserved sequences in the IVD. Additionally, a functional IVD is shown to increase the binding affinity of NEMO for IKKβ, enhance the thermal stability of NEMO, and is required to propagate NF-κB signaling in cells. A fluorescence-based assay is also developed to characterize the formation of a complex composed of NEMO, a zinc ion, and IκBα. A separate fluorescence-based assay is developed to measure IKK activity and is used to determine that NEMO alone or in the presence of linear tetraubiquitin does not enhance the rate of IKKβ phosphorylation of an IκBα-derived peptide. Furthermore, a number of organic small molecules and macrocycles are screened against the NEMO-IKKβ interaction. One small molecule was validated as an inhibitor and its biophysical properties and inhibition kinetics are described in this thesis. These analyses represent the first characterization of a highly conserved domain required for the function of the key control point in NF-κB signaling. The IVD domain of NEMO could be targeted for development of an allosteric effector for therapeutic discovery

Multiscale Simulations of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) lack stable secondary and/or tertiary structures under physiological conditions. The have now been recognized to play important roles in numerous biological processes, particularly cellular signaling and regulation. Mutation of IDPs are frequently associated with human diseases, such as cancers and neuron degenerative diseases. Therefore, it is important to understand the structure, dynamics, and interactions of IDPs, so as to establish the mechanistic basis of how intrinsic disorder mediates versatile functions and how such mechanisms may fail in human diseases. However, the heterogeneous structural ensembles of IDPs are not amenable to high resolution characterization solely through experimental measurements, and molecular modelling and simulation are required to study IDP structures, dynamics, and interactions at the atomistic levels. Here, we first applied the state-of-the-art explicit solvent atomistic simulations to an anti-apoptotic protein Bcl-xL and demonstrated how inherent structural disorder may provide a physical basis of protein regulated unfolding in signaling transduction. We have also constructed a series of efficient coarse-grained models to directly simulate the interactions between IDPs and unveiled how the preexisting structural elements accelerate binding of ACTR to NCBD by promoting efficient folding upon encounter. These studies shed important light on how IDPs perform functions in the cellular regulatory network, but also reveal the necessity of new sampling techniques for more efficient simulations of IDPs. We have thus developed a novel sampling technique, called multiscale enhanced sampling (MSES). MSES couples the atomistic model with coarse-grained ones, to accelerate the sampling of atomistic conformational space. Bias from coupling to a coarse-grained model can be removed using Hamiltonian replica exchange. To achieve the best possible efficiency of MSES simulations, we have developed a new hybrid resolution protein model that could capture the essential features of IDP structures, so as to generate local and long-range fluctuations that are largely consistent with those at the atomistic level. We have also developed an advanced replica exchange protocol, to allow the fast conformational transitions observed in the coupled conditions to be rapidly exchanged to the unbiased limit. Application of these strategies to characterize the structural ensembles of a few non-trivial IDPs shows that faster convergence rate can be achieved, demonstrating the great potential of MSES for atomistic simulations of larger and more complex IDPs

Discovery of Molecules that Modulate Protein-Protein Interactions in the Context of Human Proliferating Cell Nuclear Antigen-Associated Processes of DNA Replication and Damage Repair

Integral to cell viability is the homotrimeric protein complex Proliferating Cell Nuclear Antigen (PCNA) that encircles chromatin-bound DNA and functionally acts as a DNA clamp that provides topological sites for recruitment of proteins necessary for DNA replication and damage repair. PCNA has critical roles in the survival and proliferation of cells, as disease-associated dysregulation of associated functions can have dire effects on genome stability, leading to the formation of various malignancies ranging from non-Hodgkin’s lymphoma to skin, laryngeal, ocular, prostate and breast cancers. Here, a strategy was explored with PCNA as a drug target that may have wider implications for targeting protein-protein interactions (PPIs) as well as for fragment-based drug design. A design platform using peptidomimetic small molecules was developed that maps ideal surface binding interaction sites at a PPI interface before considering detailed conformations of an optimal ligand. A novel in silico multi-fragment, combinatorial screening approach was used to guide the selection and subsequent synthesis of tripeptoid ligands, which were evaluated in a PCNA-based competitive displacement assay. From the results, some of the peptoid-based compounds that were synthesized displayed the ability to disrupt the interaction between PCNA and a PIP box-containing peptide. The IC50 values of these compounds had similar or improved affinity to that of T2AA, an established inhibitor of PCNA-PIP box interactions. The information gained here could be useful for subsequent drug lead candidate identification