Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic-aromatic interactions

Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional π-systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic-aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic-aromatic interactions at this interface suggested that substitution of residue TyrB26 by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [TrpB26]insulin analog (determined as a T3Rf3 zinc hexamer at a resolution of 2.25 Å) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the in vitro lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the TrpB26-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly

EXPERIMENTAL AND MOLECULAR DYNAMICS SIMULATION STUDIES OF PARTITIONING AND TRANSPORT ACROSS LIPID BILAYER MEMBRANES

Most drugs undergo passive transport during absorption and distribution in the body. It is desirable to predict passive permeation of future drug candidates in order to increase the productivity of the drug discovery process. Unlike drug-receptor interactions, there is no receptor map for passive permeability because the process of transport across the lipid bilayer involves multiple mechanisms. This work intends to increase the understanding of permeation of drug-like molecules through lipid bilayers. Drug molecules in solution typically form various species due to ionization, complexation, etc. Therefore, species specific properties must be obtained to bridge the experiment and simulations. Due to the volume contrast between intra- and extravesicular compartments of liposomes, minor perturbations in ionic and binding equilibria become significant contributors to transport rates. Using tyramine as a model amine, quantitative numerical models were developed to determine intrinsic permeability coefficients. The microscopic ionization and binding constants needed for this were independently measured. The partition coefficient in 1,9-decadiene was measured for a series of compounds as a quantitative surrogate for the partitioning into the hydrocarbon region of the bilayer. These studies uncovered an apparent long-range interaction between the two polar substituents that caused deviations in the microscopic pKa values and partition coefficient of tyramine from the expected values. Additionally the partition coefficients in the preferred binding region of the bilayer were also measured by equilibrium uptake into liposomes. All-atom molecular dynamics simulations of lipid bilayers containing tyramine, 4- ethylphenol, or phenylethylamine provided free energies of transfer of these solutes from water to various locations on the transport path. The experimentally measured partition coefficients were consistent with the free energy profiles in showing the barrier in the hydrocarbon region and preferred binding region near the interface. The substituent contributions to these free energies were also quantitatively consistent between the experiments and simulations. Specific interactions between solutes and the bilayer suggest that amphiphiles are likely to show preferred binding in the head group region and that the most of hydrogen bonds involving solutes located inside the bilayer are with water molecules. Solute re-orientation inside the bilayer lowers the partitioning barrier by allowing favorable interactions

Evaluating Force Fields for the Computational Prediction of Ionized Arginine and Lysine Side-Chains Partitioning into Lipid Bilayers and Octanol

Abundant peptides and proteins containing arginine (Arg) and lysine (Lys) amino acids can apparently permeate cell membranes with ease. However, the mechanisms by which these peptides and proteins succeed in traversing the free energy barrier imposed by cell membranes remain largely unestablished. Precise thermodynamic studies (both theoretical and experimental) on the interactions of Arg and Lys residues with model lipid bilayers can provide valuable clues to the efficacy of these cationic peptides and proteins. We have carried out molecular dynamics simulations to calculate the interactions of ionized Arg and Lys side-chains with the zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayer for 10 widely used lipid/protein force fields: CHARMM36/CHARMM36, SLIPID/AMBER99SB-ILDN, OPLS-AA/OPLS-AA, Berger/OPLS-AA, Berger/GROMOS87, Berger/GROMOS53A6, GROMOS53A6/GROMOS53A6, nonpolarizable MARTINI, polarizable MARTINI, and BMW MARTINI. We performed umbrella sampling simulations to obtain the potential of mean force for Arg and Lys side-chains partitioning from water to the bilayer interior. We found significant differences between the force fields, both for the interactions between side-chains and bilayer surface, as well as the free energy cost for placing the side-chain at the center of the bilayer. These simulation results were compared with the WimleyWhite interfacial scale. We also calculated the free energy cost for transferring ionized Arg and Lys side-chains from water to both dry and wet octanol. Our simulations reveal rapid diffusion of water molecules into octanol whereby the equilibrium mole fraction of water in the wet octanol phase was similar to 25%. Surprisingly, our free energy calculations found that the high water content in wet octanol lowered the water-to-octanol partitioning free energies for cationic residues by only 0.6 to 0.7 kcal/mol

Molecular Mechanisms of Resistance and Structure-Based Drug Design in Homodimeric Viral Proteases

Drug resistance is a global health threat costing society billions of dollars and impacting millions of lives each year. Current drug design strategies are inadequate because they focus on disrupting target activity and not restricting the evolutionary pathways to resistance. Improved strategies would exploit the structural and dynamic changes in the enzyme–inhibitor system integrating data from many inhibitors and variants. Using HIV-1 protease as a model system, I aimed to elucidate the underlying resistance mechanisms, characterize conserved protease-inhibitor interactions, and generate more robust inhibitors by applying these insights. For primary mechanisms of resistance, comparing interactions at the protease–inhibitor interface showed how specific modifications affected potency. For mutations distal to the active site, molecular dynamics simulations were necessary to elucidate how changes propagated to reduce inhibitor binding. These insights informed inhibitor design to improve potency against highly resistant variants by optimizing hydrogen bonding. A series of hybrid inhibitors was also designed that showed excellent potency by combining key moieties of multiple FDA-approved inhibitors. I characterized the structural basis for alterations in binding affinity in HIV-1 protease both from mutations and inhibitors. I applied these strategies to HTLV-1 protease, a potential drug target. I identified the HIV-1 inhibitor darunavir as a viable scaffold and evaluated analogues, leading to a low-nanomolar compound with potential for optimization. Hopefully, insights from this thesis will lead to the development of potent HTLV-1 protease inhibitors. More broadly, these inhibitor design strategies are applicable to other rapidly evolving targets, thereby reducing drug resistance rates in the future

X-Ray Crystal Structures And Enzyme Kinetic Investigations Of Drug-Resistant Mutants Of Hiv-1 Protease

Globally, 62% of 38 million HIV-infected people are receiving antiretroviral therapy. Inhibitors targeting the viral protease have been clinically successful as 9 protease inhibitors (PIs) have been approved by the FDA since 1995. However, drug resistance arising by mutations in the protease undermines effective treatment. Analysis of protease and its mutants by structural biology methods paired with enzymology has given insight into the molecular mechanisms for drug resistance and guided new strategies for inhibitor design. Recently, highly resistant variants of HIV-1 protease from clinical isolates have been identified with ~20 mutations and several orders of magnitude worse binding affinity for clinical PIs such as darunavir. Three such mutants, PR20, PRS17, and PRS5B, are the focus of this body of work and show 800-10,000-fold less susceptibility to darunavir than wild-type protease. Understanding the molecular mechanisms driving the extreme drug-resistance of these three protease mutants aid rational drug design efforts to fight the HIV/AIDS pandemic. Structure-guided strategies for drug design have resulted in an investigational inhibitor, GRL-142, with modified ligands derived from the darunavir scaffold that shows 16-fold better inhibition than darunavir of resistant mutant PR20. The crystal structure of PR20 in complex with GRL-142 reveals how the expanded binding pocket, dynamic flaps, and faster dimer dissociation of PR20 are counteracted by the larger moieties of GRL-142. Resistant variant PRS17, which was rationally selected from the HIVdb genotype-phenotype database by machine learning, shows ~3-fold better inhibition by peptide substrate analogs compared to wild-type protease. Crystal structures of PRS17 with substrate analogs show a major effect of drug-resistance mutations V82S and G48V improving interactions with substrates consistent with better inhibition, suggesting a novel mechanism for resistance. Finally, structural studies of another mutant selected by machine learning, PRS5B, reveal coordinated structural changes leading to decreased intra-subunit interactions and intermediate levels of resistance to PIs. The sum of knowledge on resistant variants PR20, PRS17, and PRS5B illuminates the evolution of HIV-1 protease in the era of accessible PI treatments. These results illustrate the power of combining structural analysis of proteins with enzyme kinetics for combatting drug resistant HIV

Molecular Simulation Studies of Hydrophobins near Gas, Oil and Water Interfaces

This dissertation contains fundamental, classical molecular simulation studies of the properties of hydrophobins (a unique family of surface-active proteins produced by filamentous fungi in soil) near interfaces involving gas, oil, water, organic solvents and polymers. These studies are relevant to possibly using hydrophobins as natural oil dispersants and in processing of polymers. Preliminary studies by the Russo group suggest that these small surface-active proteins can encapsulate oil, gases and polymers in cylindrical structures. We have performed classic molecular dynamics (MD) simulation and potential of mean force (PMF) calculations of a class I hydrophobin EAS using both all-atom and coarse-grained representations. The interfacial properties of these hydrophobins at gas/water and oil/water interfaces were probed, and our simulation results qualitatively agree with experimental observations. According to the PMF calculation results, EAS molecule was likely to stay at the hydrophobic/hydrophilic interface, and the adsorption behavior of EAS at the interface was strong and irreversible. We performed MD simulations using Martini coarse-grained (CG) models to gain insight into the stability of nm-sized ‘blobs’ formed by the assembly of hydrophobin around oil. Finally, we developed a Martini model for Poly(ã-stearyl á,L-glutamate), PSLG, and used it to perform MD simulations of PSLG molecules near a hydrophobin-coated hydrophilic-hydrophobic interface. These simulations suggest that initial alignment of the PSLG chains, PSLG concentration and solvent type may affect the final alignment of the PSLG chains

Exploring the Role of Large Clusters of Branched Aliphatic Residues on the Folding Free Energy Landscape of (βα)8 TIM Barrel Proteins

(βα)8 TIM barrel proteins are one of the most common structural motifs found in biology. They have a complex folding free energy landscape that includes an initial off-pathway intermediate as well as two on-pathway intermediates. The formation of these intermediates is hypothesized to be driven by large clusters of the branched chain amino acids, isoleucine, leucine, and valine (ILV). All-atom MD simulations and circular dichroism experiments on polar mutants of the hydrophobic clusters of α-Trp synthase, a TIM barrel protein, revealed the importance of dehydrating the clusters on intermediate states. Custom, single-piece microfluidic chips were interfaced with small angle x-ray scattering and time resolved FRET experiments to monitor the role of a large ILV cluster on the microsecond timescale in a second TIM barrel protein, sIGPS. Dimensional analysis of the initial misfolded intermediate showed an ILV cluster was responsible for the initiation of structure in the intermediate. Early structure formation in the ILV cluster was confirmed by coarse grained simulations. Native state hydrogen exchange experiments were used to probe the higher energy species that are in equilibrium with the native state. Results from the NMR experiment complement the kinetic studies as the core of stability found by NMR mapped back to the same region of the ILV cluster that was found to initiate folding. When taken together, the results show the importance of hydrophobic clusters on the entire free energy surface of TIM barrel proteins

Membrane bending is critical for assessing the thermodynamic stability of proteins in the membrane

The ability of biological membranes to bend is critical to understanding the interaction between proteins and the lipid bilayer. Experimental and computational studies have shown that the membrane can bend to expose charged and polar residues to the lipid headgroups and water, greatly reducing the cost of protein insertion. However, current computational approaches are poorly equipped to accurately model such deformation; atomistic simulations often do not reach the time-scale necessary to observe large-scale rearrangement, and continuum approaches assume a flat, rigid bilayer. In this thesis we present an efficient computational model of a deformable membrane for probing these interactions with elasticity theory and continuum electrostatics. To validate the model, we first investigate the insertion of three membrane proteins and three aqueous proteins. The model finds the membrane proteins and aqueous proteins stable and unstable in the membrane, respectively. We also investigate the sensitivity of these predictions to changes in several key parameters. The model is then applied to interactions between the membrane and the voltage sensor segments of voltage-gated potassium channels. Despite their high numbers of basic residues, experiments have shown that voltage sensors can be stably accommodated in the membrane. For simple continuum electrostatics approaches that assume a flat membrane, the penalty of inserting these charged residues would seem to prohibit voltage sensor insertion. However, in our method the membrane deforms to enable interaction between solvent and the charged residues. Our calculations predict that the highly charged S4 helices of several potassium channels are in fact stable in the membrane, in accord with experimental observations. Experimental and computational evidence has shown that the cost for inserting multiple charged amino acids into the membrane is not additive; it is not as costly to insert a second charge once a first has already been inserted. Our model reflects this phenomenon and provides a simple mechanical explanation linked to membrane deformation. We additionally consider the energetics of passive ion penetration into the membrane from bulk solvent. We use coarse-grained molecular dynamics to guide our input parameters and show that ion permeation energy profiles agree with atomistic simulations when membrane bending is included

Solvation thermodynamics of organic molecules by the molecular integral equation theory : approaching chemical accuracy

The integral equation theory (IET) of molecular liquids has been an active area of academic research in theoretical and computational physical chemistry for over 40 years because it provides a consistent theoretical framework to describe the structural and thermodynamic properties of liquid-phase solutions. The theory can describe pure and mixed solvent systems (including anisotropic and nonequilibrium systems) and has already been used for theoretical studies of a vast range of problems in chemical physics / physical chemistry, molecular biology, colloids, soft matter, and electrochemistry. A consider- able advantage of IET is that it can be used to study speci fi c solute − solvent interactions, unlike continuum solvent models, but yet it requires considerably less computational expense than explicit solvent simulations