ß-Branched Amino Acids Stabilize Specific Conformations of Cyclic Hexapeptides.

Cyclic peptides (CPs) are a promising class of molecules for drug development, particularly as inhibitors of protein-protein interactions. Predicting low-energy structures and global structural ensembles of individual CPs is critical for the design of bioactive molecules, but these are challenging to predict and difficult to verify experimentally. In our previous work, we used explicit-solvent molecular dynamics simulations with enhanced sampling methods to predict the global structural ensembles of cyclic hexapeptides containing different permutations of glycine, alanine, and valine. One peptide, cyclo-(VVGGVG) or P7, was predicted to be unusually well structured. In this work, we synthesized P7, along with a less well-structured control peptide, cyclo-(VVGVGG) or P6, and characterized their global structural ensembles in water using NMR spectroscopy. The NMR data revealed a structural ensemble similar to the prediction for P7 and showed that P6 was indeed much less well-structured than P7. We then simulated and experimentally characterized the global structural ensembles of several P7 analogs and discovered that ß-branching at one critical position within P7 is important for overall structural stability. The simulations allowed deconvolution of thermodynamic factors that underlie this structural stabilization. Overall, the excellent correlation between simulation and experimental data indicates that our simulation platform will be a promising approach for designing well-structured CPs and also for understanding the complex interactions that control the conformations of constrained peptides and other macrocycles.

Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

The acetylcholine-activated inward rectifier potassium current ( IKACh) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that the blocking of IKACh with the small molecule chloroquine terminates persistent AF. We used a sheep model of tachypacing-induced, persistent AF, molecular modeling, electrophysiology, and structural biology approaches. The 50% inhibition/inhibitory concentration of IKACh block with chloroquine, measured by patch clamp, was 1 µM. In optical mapping of sheep hearts with persistent AF, 1 µM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of IKACh) by interacting with residues D260 and F255, in proximity to I228, Q227, and L299. 1H 15N heteronuclear single-quantum correlation of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical-shift perturbations upon drug binding. We then crystallized and solved a 2.5 Å X-ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drug's binding to the pore becomes off centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A and D260A mutations significantly reduced IKACh block by chloroquine. With the use of numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel and exert antiarrhythmic effects. Chloroquine binds the IKACh channel at a site formed by specific amino acids in the ion-permeation pathway, leading to decreased IKACh and the subsequent termination of AF.-Takemoto, Y., Slough, D. P., Meinke, G., Katnik, C., Graziano, Z. A., Chidipi, B., Reiser, M., Alhadidy, M. M., Ramirez, R., Salvador-Montañés, O., Ennis, S., Guerrero-Serna, G., Haburcak, M., Diehl, C., Cuevas, J., Jalife, J., Bohm, A., Lin,Y.-S., Noujaim, S. F. Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

Understanding and designing head-to-tail cyclic peptides.

Cyclic peptides (CPs) are an exciting class of molecules with a variety of applications. However, design strategies for CP therapeutics, for example, are generally limited by a poor understanding of their sequence-structure relationships. This knowledge gap often leads to a trial-and-error approach for designing CPs for a specific purpose, which is both costly and time-consuming. Herein, we describe the current experimental and computational efforts in understanding and designing head-to-tail CPs along with their respective challenges. In addition, we provide several future directions in the field of computational CP design to improve its accuracy, efficiency and applicability. These advances, combined with experimental techniques, shall ultimately provide a better understanding of these interesting molecules and a reliable working platform to rationally design CPs with desired characteristics.

Toward accurately modeling N-methylated cyclic peptides.

Cyclic peptides have unique properties and can target protein surfaces specifically and potently. N-Methylation provides a promising way to further optimize the pharmacokinetic and structural profiles of cyclic peptides. The capability to accurately model structures adopted by N-methylated cyclic peptides would facilitate rational design of this interesting and useful class of molecules. We apply molecular dynamics simulations with advanced enhanced sampling methods to efficiently characterize the structural ensembles of N-methylated cyclic peptides, while simultaneously evaluating the overall performance of several simulation force fields. We find that one of the residue-specific force fields, RSFF2, is able to recapitulate experimental structures of the N-methylated cyclic peptide benchmarks tested here when the correct amide isomers are used as initial configurations and enforced during the simulations. Thus, using our simulation approach, it is possible to accurately and efficiently predict the structures of N-methylated cyclic peptides if sufficient information is available to determine the correct amide cis/trans configuration. Moreover, our results suggest that, upon further optimization of RSFF2 to more reliably predict cis/trans isomers, molecular dynamics simulations will be able to de novo predict N-methylated cyclic peptides in the near future, strongly motivating such continued optimization.

Mapping the sequence-structure relationships of simple cyclic hexapeptides.

Cyclic peptides are promising protein-protein interaction modulators with high binding affinities and specificities, as well as enhanced stabilities and oral availabilities over linear analogs. Despite their relatively small size and cyclic architecture, it is currently difficult to predict the favored conformation(s) of most classes of cyclic peptides. An improved understanding of the sequence-structure relationships for cyclic peptides will offer an avenue for the rational design of cyclic peptides as possible therapeutics. In this work, we systematically explored the sequence-structure relationships for two cyclic hexapeptide systems using molecular dynamics simulation techniques. Starting with an all-glycine cyclic hexapeptide, cyclo-G6, we systematically replaced glycine residues with alanines and characterized the structural ensembles of different variants. The same process was repeated with valines to investigate the effects of larger side chains. An analysis of the origin of structure preferences was performed using thermodynamics decomposition and several general observations are reported.

The Antimalarial Chloroquine Reduces the Burden of Persistent Atrial Fibrillation.

In clinical practice, reducing the burden of persistent atrial fibrillation by pharmacological means is challenging. We explored if blocking the background and the acetylcholine-activated inward rectifier potassium currents (IK1 and IKACh) could be antiarrhythmic in persistent atrial fibrillation. We thus tested the hypothesis that blocking IK1 and IKACh with chloroquine decreases the burden of persistent atrial fibrillation. We used patch clamp to determine the IC50 of IK1 and IKACh block by chloroquine and molecular modeling to simulate the interaction between chloroquine and Kir2.1 and Kir3.1, the molecular correlates of IK1 and IKACh. We then tested, as a proof of concept, if oral chloroquine administration to a patient with persistent atrial fibrillation can decrease the arrhythmia burden. We also simulated the effects of chloroquine in a 3D model of human atria with persistent atrial fibrillation. In patch clamp the IC50 of IK1 block by chloroquine was similar to that of IKACh. A 14-day regimen of oral chloroquine significantly decreased the burden of persistent atrial fibrillation in a patient. Mathematical simulations of persistent atrial fibrillation in a 3D model of human atria suggested that chloroquine prolonged the action potential duration, leading to failure of reentrant excitation, and the subsequent termination of the arrhythmia. The combined block of IK1 and IKACh can be a targeted therapeutic strategy for persistent atrial fibrillation.

Designing Well-Structured Cyclic Pentapeptides Based on Sequence-Structure Relationships.

Cyclic peptides are a promising class of molecules for unique applications. Unfortunately, cyclic peptide design is severely limited by the difficulty in predicting the conformations they will adopt in solution. In this work, we use explicit-solvent molecular dynamics simulations to design well-structured cyclic peptides by studying their sequence-structure relationships. Critical to our approach is an enhanced sampling method that exploits the essential transitional motions of cyclic peptides to efficiently sample their conformational space. We simulated a range of cyclic pentapeptides from all-glycine to a library of cyclo-(X1X2AAA) peptides to map their conformational space and determine cooperative effects of neighboring residues. By combining the results from all cyclo-(X1X2AAA) peptides, we developed a scoring function to predict the structural preferences for X1-X2 residues within cyclic pentapeptides. Using this scoring function, we designed a cyclic pentapeptide, cyclo-(GNSRV), predicted to be well structured in aqueous solution. Subsequent circular dichroism and NMR spectroscopy revealed that this cyclic pentapeptide is indeed well structured in water, with a nuclear Overhauser effect and J-coupling values consistent with the predicted structure.

Correlated rotational switching in two-dimensional self-assembled molecular rotor arrays.

Molecular devices are capable of performing a number of functions from mechanical motion to simple computation. Their utility is somewhat limited, however, by difficulties associated with coupling them with either each other or with interfaces such as electrodes. Self-assembly of coupled molecular devices provides an option for the construction of larger entities that can more easily integrate with existing technologies. Here we demonstrate that ordered organometallic arrays can be formed spontaneously by reaction of precursor molecular rotor molecules with a metal surface. Scanning tunnelling microscopy enables individual rotors in the arrays to be switched and the resultant switches in neighbouring rotors imaged. The structure and dimensions of the ordered molecular rotor arrays dictate the correlated switching properties of the internal submolecular rotor units. Our results indicate that self-assembly of two-dimensional rotor crystals produces systems with correlated dynamics that would not have been predicted a priori.