Huntington’s disease (HD) is fatal neurodegenerative disorder caused by the expansion of a CAG-repeat tract in the huntingtin ( HTT ) gene. Human and mouse genetics studies have demonstrated a role for DNA mismatch repair (MMR) proteins which control the rate of somatic expansion of the HTT CAG repeat and disease onset and progression. MutSβ, a key member of the MMR pathway, is a heterodimeric protein of MSH2 and MSH3 that recognizes and initiates the repair of small insertion or deletion DNA loop outs. Both mouse Msh3 loss-of-function and reduced-expression alleles of human MSH3 lead to slower rates of somatic expansion in the HTT CAG tract and a delay of disease onset and progression, signifying MSH3 as a promising drug target for HD. Structural biology studies of MutSβ are informative for mechanism, protein structure-function relationships, and guiding small-molecule drug design. Here we report biochemical and cryo-electron microscopy analyses of human MutSβ ensembles, revealing that MutSβ undergoes multiple conformational changes in response to binding and release of nucleotides and DNA. The DNA-free MutSβ-ADP complex adopts an open conformation that is compatible with DNA binding. The conformation of MutSβ in the (CAG) 2 DNA-bound open structure most closely resembles the recently identified low-affinity state of MutSα, compared to the canonical mismatch-bound conformation. The homoduplex-bound and DNA-unbound MutSβ-ATP structures show that MutSβ undergoes an ATP-dependent conformational change towards sliding clamp forms. This study provides a comprehensive understanding of the structural conformational dynamics of MutSβ, insights into the MMR cascade, and a foundation for structure-guided drug discovery.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00344.].

Objectives The purpose of this article is to introduce an emerging field called ‘Biopharmaceutical Informatics’. It describes how tools from Information technology and Molecular Biophysics can be adapted, developed and gainfully employed in discovery and development of biologic drugs. Key Findings The findings described here are based on literature surveys and the authors’ collective experiences in the field of biologic drug product development. A strategic framework to forecast early the hurdles faced during drug product development is weaved together and elucidated using chemical degradation as an example. Efficiency of translating biologic drug discoveries into drug products can be significantly improved by combining learnings from experimental biophysical and analytical data on the drug candidates with molecular properties computed from their sequences and structures via molecular modeling and simulations. Summary Biopharmaceutical Informatics seeks to promote applications of computational tools towards discovery and development of biologic drugs. When fully implemented, industry-wide, it will enable rapid materials-free developability assessments of biologic drug candidates at early stages as well as streamline drug product development activities such as commercial scale production, purification, formulation, analytical characterization, safety and in vivo performance.

Early identification of asparagine deamidation and aspartate isomerization degradation sites can facilitate the successful development of biopharmaceuticals. Several knowledge-based models have been proposed to assess these degradation risks. In this study, we propose a physics-based approach to identify the degradation sites on the basis of the free-energy barriers along the prechemical conformational step and the chemical reaction pathway. These contributions are estimated from classical and quantum mechanics/molecular mechanics molecular dynamics simulations. The computed barriers are compared to those for reference reactions in water within GNG and GDG sequence motifs in peptides (which demonstrate the highest degradation rates). Two major factors decreasing the degradation rates relative to the reference reactions are steric hindrance toward accessing reactive conformations and replacement of water by less polar side chains in the solvation shell of transition states. Among the potential degradation sites in the complementarity-determining region of trastuzumab and between two DK sites in glial cell-derived neurotropic factor, this method identified N30T, N55G, D102G, and D95K, respectively, in agreement with experiments. This approach can be incorporated in early computational screening of chemical degradation sites in biopharmaceuticals.

The observed pressure sensitivity of the isomerization reaction rate of bis-anthracene cyclophane photoisomer has attracted significant attention in the rational design of mechanically sensitive materials. However, the molecular origin of this sensitivity remains unclear. We developed an ab initio molecular model to quantify the effect of pressure on the reaction rate and to elucidate its molecular origin. Pressure-induced deformations and changes along the reaction free-energy surfaces are estimated from ab initio molecular dynamics trajectories. Our model predicts a barrier reduction of similar to 2 kcal/mol at 0.9 GPa (in agreement with experiment). The barrier reduction is linear in the low-pressure regime (up to 2 GPa) but has a nonlinear dependence at higher pressures. We find that pressure alters the reaction path and that the mechanical sensitivity of the reaction rate is caused by an uneven distribution of the free-energy increase along the reaction surface. The uneven distribution primarily results from destabilization of the reactant, which has a lower mechanical rigidity along a particular deformation mode (flattening of anthracene rings as they are pushed toward each other in the cyclophane framework). Exploiting similar structural motifs to maximize rigidity differences along the reaction coordinate represents a promising rational design strategy.

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTPressure-Induced Neutral-to-Ionic Transition in an Amorphous Organic MaterialYi Ren†, Semin Lee‡, James M. Christensen†, Nikolay V. Plotnikov§, Mark Burgess†, Todd J. Martínez§, Dana D. Dlott†, and Jeffrey S. Moore*†‡View Author Information† Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States‡ Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States§ Department of Chemistry, Stanford University, Stanford, California 94305, United States*Jeffrey S. Moore. Email: [email protected]Cite this: Chem. Mater. 2016, 28, 18, 6446–6449Publication Date (Web):September 13, 2016Publication History Received3 July 2016Revised4 September 2016Published online16 September 2016Published inissue 27 September 2016https://pubs.acs.org/doi/10.1021/acs.chemmater.6b02703https://doi.org/10.1021/acs.chemmater.6b02703rapid-communicationACS PublicationsCopyright © 2016 American Chemical SocietyRequest reuse permissionsArticle Views1151Altmetric-Citations4LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Absorption,Amorphous materials,Charge transfer,Materials,Polymers Get e-Alerts

Proposed in this contribution is a protocol for calculating fine-physics (e.g., ab initio QM/MM) free-energy surfaces at a high level of accuracy locally (e.g., only at reactants and at the transition state for computing the activation barrier) from targeted fine-physics sampling and extensive exploratory coarse-physics sampling. The full free-energy surface is still computed but at a lower level of accuracy from coarse-physics sampling. The method is analytically derived in terms of the umbrella sampling and the free-energy perturbation methods which are combined with the thermodynamic cycle and the targeted sampling strategy of the paradynamics approach. The algorithm starts by computing low-accuracy fine-physics free-energy surfaces from the coarse-physics sampling in order to identify the reaction path and to select regions for targeted sampling. Thus, the algorithm does not rely on the coarse-physics minimum free-energy reaction path. Next, segments of high-accuracy free-energy surface are computed locally at selected regions from the targeted fine-physics sampling and are positioned relative to the coarse-physics free-energy shifts. The positioning is done by averaging the free-energy perturbations computed with multistep linear response approximation method. This method is analytically shown to provide results of the thermodynamic integration and the free-energy interpolation methods, while being extremely simple in implementation. Incorporating the metadynamics sampling to the algorithm is also briefly outlined. The application is demonstrated by calculating the B3LYP//6-31G*/MM free-energy barrier for an enzymatic reaction using a semiempirical PM6/MM reference potential. These modifications allow computing the activation free energies at a significantly reduced computational cost but at the same level of accuracy compared to computing full potential of mean force.

GTPases play a major role in cellular processes, and gaining quantitative understanding of their activation demands reliable free energy surfaces of the relevant mechanistic paths in solution, as well as the interpolation of this information to GTPases. Recently, we generated ab initio quantum mechanical/molecular mechanical free energy surfaces for the hydrolysis of phosphate monoesters in solution, establishing quantitatively that the barrier for the reactions with a proton transfer (PT) step from a single attacking water (1W) is higher than the one where the PT is assisted by a second water (2W). The implication of this finding on the activation of GTPases is quantified here, by using the ab initio solution surfaces to calibrate empirical valence bond surfaces and then exploring the origin of the activation effect. It is found that, although the 2W PT path is a new element, this step is not rate determining, and the catalytic effect is actually due to the electrostatic stabilization of the pre-PT transition state and the subsequent plateau. Thus, the electrostatic catalytic effect found in our previous studies of the Ras GTPase activating protein (RasGAP) and the elongation factor-Tu (EF-Tu) with a 1W mechanism is still valid for the 2W path. Furthermore, as found before, the corresponding activation appears to involve a major allosteric effect. Overall, we believe that our finding is general to both GTPases and ATPases. In addition to the biologically relevant finding, we also provide a critical discussion of the requirements from reliable surfaces for enzymatic reactions.

Understanding the nature of the free-energy surfaces for phosphate hydrolysis is a prerequisite for understanding the corresponding key chemical reactions in biology. Here, the challenge has been to move to careful ab initio QM/MM (QM(ai)/MM) free-energy calculations, where obtaining converging results is very demanding and computationally expensive. This work describes such calculations, focusing on the free-energy surface for the hydrolysis of phosphate monoesters, paying special attention to the comparison between the one water (1W) and two water (2W) paths for the proton-transfer (PT) step. This issue has been explored before by energy minimization with implicit solvent models and by nonsystematic QM/MM energy minimization, as well as by nonsystematic free-energy mapping. However, no study has provided the needed reliable 2D (3D) surfaces that are necessary for reaching concrete conclusions. Here we report a systematic evaluation of the 2D (3D) free-energy maps for several relevant systems, comparing the results of QM(ai)/MM and QM(ai)/implicit solvent surfaces, and provide an advanced description of the relevant energetics. It is found that the 1W path for the hydrolysis of the methyl diphosphate (MDP) trianion is 6-9 kcal/mol higher than that the 2W path. This difference becomes slightly larger in the presence of the Mg(2+) ion because this ion reduces the pKa of the conjugated acid form of the phosphate oxygen that accepts the proton. Interestingly, the BLYP approach (which has been used extensively in some studies) gives a much smaller difference between the 1W and 2W activation barriers. At any rate, it is worth pointing out that the 2W transition state for the PT is not much higher that the common plateau that serves as the starting point of both the 1W and 2W PT paths. Thus, the calculated catalytic effects of proteins based on the 2W PT mechanistic model are not expected to be different from the catalytic effects predicted using the 1W PT mechanistic model, which was calibrated on the observed barrier in solution and in which the TS charge distribution was similar to the that of the plateau (as was done in all of our previous EVB studies).

The nature and mechanism of phosphate hydrolysis reactions are of great interest in view of the crucial role of these reactions in key biological processes. Although it is becoming clearer that the ultimate way of resolving mechanistic controversies must involve reliable theoretical studies, it is not widely realized that such studies cannot be performed at present by using most existing automated ways and that only careful systematic studies can lead to meaningful conclusions. The present work clarifies the above point by considering the hydrolysis of phosphate monoesters. The clarification starts by defining the actual issues that should be addressed in careful studies and by highlighting the problems with studies that ignore the need for unique mechanistic definitions (e.g., works that confuse associative and dissociative pathways). We then focus on the analysis of the proton transfer (PT) pathways in phosphate hydrolysis and on recent suggestions that PT involves more than one water molecule. Here we point out that most of the studies that found a proton transfer through several water molecules have not involved a sufficient systematic search of the relevant reaction coordinates. This includes both energy minimization approaches as well as a recent metadynamics (MTD) simulation study. To illustrate the crucial need of exploring the potential surfaces reliably, rather than relying on automated approaches, we present here a very careful study of the free energy landscape along a 3D reaction coordinate (RC) exploring both the standard 2D RC, comprised of the attacking and leaving group reaction coordinates, as well as of the proton transfer (PT) coordinate. Our study points out that QM/MM minimization or MTD studies that concluded that the hydrolysis of phosphate monoesters involves a PT through several water molecules, have not explored carefully the single water (1W) path (that involves a direct PT form the attacking water molecule to the phosphate oxygen). Furthermore, we identified the most likely reason for the difficulty in finding the 1W path by QM/MM minimization methods, as well as by the current MTD simulations. We also discuss the problems with current studies that challenge the phosphate as a base mechanism and emphasize that all recent studies found associative/concerted paths (although many have not realized the meaning of their results). Finally, although we clearly do not have the last word about the 1W versus 2W paths we believe that we illustrated that the crucial mechanistic problems with alternative pathways should not be resolved by just running black box search approaches.