Fragments and hot spots in drug discovery

R01 GM064700 - NIGMS NIH HHSPublished versio

How proteins bind macrocycles

The potential utility of synthetic macrocycles (MCs) as drugs, particularly against low-druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of MCs for good target protein-binding activity or bioavailability. To address this knowledge gap, we analyze the binding modes of a representative set of MC-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic MC libraries with structural and physicochemical features likely to favor strong binding to protein targets as well as good bioavailability. We additionally provide evidence that large, natural product-derived MCs can bind targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product-inspired synthetic MCs can expand the number of proteins that are druggable by synthetic small molecules.R01 GM094551 - NIGMS NIH HHS; GM064700 - NIGMS NIH HHS; GM094551 - NIGMS NIH HHS; R01 GM064700 - NIGMS NIH HHS; GM094551-01S1 - NIGMS NIH HH

Efficient maintenance and update of nonbonded lists in macromolecular simulations

Molecular mechanics and dynamics simulations use distance based cutoff approximations for faster computation of pairwise van der Waals and electrostatic energy terms. These approximations traditionally use a precalculated and periodically updated list of interacting atom pairs, known as the “nonbonded neighborhood lists” or nblists, in order to reduce the overhead of finding atom pairs that are within distance cutoff. The size of nblists grows linearly with the number of atoms in the system and superlinearly with the distance cutoff, and as a result, they require significant amount of memory for large molecular systems. The high space usage leads to poor cache performance, which slows computation for large distance cutoffs. Also, the high cost of updates means that one cannot afford to keep the data structure always synchronized with the configuration of the molecules when efficiency is at stake. We propose a dynamic octree data structure for implicit maintenance of nblists using space linear in the number of atoms but independent of the distance cutoff. The list can be updated very efficiently as the coordinates of atoms change during the simulation. Unlike explicit nblists, a single octree works for all distance cutoffs. In addition, octree is a cache-friendly data structure, and hence, it is less prone to cache miss slowdowns on modern memory hierarchies than nblists. Octrees use almost 2 orders of magnitude less memory, which is crucial for simulation of large systems, and while they are comparable in performance to nblists when the distance cutoff is small, they outperform nblists for larger systems and large cutoffs. Our tests show that octree implementation is approximately 1.5 times faster in practical use case scenarios as compared to nblists

Improved Modeling of Peptide-Protein Binding Through Global Docking and Accelerated Molecular Dynamics Simulations

This work is licensed under a Creative Commons Attribution 4.0 International License.Peptides mediate up to 40% of known protein-protein interactions in higher eukaryotes and play a key role in cellular signaling, protein trafficking, immunology, and oncology. However, it is challenging to predict peptide-protein binding with conventional computational modeling approaches, due to slow dynamics and high peptide flexibility. Here, we present a prototype of the approach which combines global peptide docking using ClusPro PeptiDock and all-atom enhanced simulations using Gaussian accelerated molecular dynamics (GaMD). For three distinct model peptides, the lowest backbone root-mean-square deviations (RMSDs) of their bound conformations relative to X-ray structures obtained from PeptiDock were 3.3–4.8 Å, being medium quality predictions according to the Critical Assessment of PRediction of Interactions (CAPRI) criteria. GaMD simulations refined the peptide-protein complex structures with significantly reduced peptide backbone RMSDs of 0.6–2.7 Å, yielding two high quality (sub-angstrom) and one medium quality models. Furthermore, the GaMD simulations identified important low-energy conformational states and revealed the mechanism of peptide binding to the target proteins. Therefore, PeptiDock+GaMD is a promising approach for exploring peptide-protein interactions

Protein docking refinement by convex underestimation in the low-dimensional subspace of encounter complexes

We propose a novel stochastic global optimization algorithm with applications to the refinement stage of protein docking prediction methods. Our approach can process conformations sampled from multiple clusters, each roughly corresponding to a different binding energy funnel. These clusters are obtained using a density-based clustering method. In each cluster, we identify a smooth “permissive” subspace which avoids high-energy barriers and then underestimate the binding energy function using general convex polynomials in this subspace. We use the underestimator to bias sampling towards its global minimum. Sampling and subspace underestimation are repeated several times and the conformations sampled at the last iteration form a refined ensemble. We report computational results on a comprehensive benchmark of 224 protein complexes, establishing that our refined ensemble significantly improves the quality of the conformations of the original set given to the algorithm. We also devise a method to enhance the ensemble from which near-native models are selected.Published versio

Analysis of Binding Site Hot Spots on the Surface of Ras GTPase

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the “off” and “on” allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target

Encounter complexes and dimensionality reduction in protein-protein association

An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition

Вплив модифікованої плікації шлунка та рукавної резекції шлунка у щурів із глутамат-індукованим ожирінням та стрептозотоцин- індукованим діабетом на динаміку зниження маси тіла та вуглеводний обмін

The aim of the work: to compare the influence of modified gastric plication and gastric resection in rats with glutamate-induced obe­sity and streptozotocin-induced diabetes on the dynamics of body weight and hydrocarbon metabolism.Materials and Methods. The study was probed on 45 white, non-breeding female rats with an initial body weight of 150–180 grams. Glutamate-induced obesity and streptotrozine-induced diabetes were modeled on animals. Animals were divided into 3 groups with 15 animals in each control group, and two experimental groups. In the first experimental group we performed sleeve resection of the stomach; in the second one plication of the stomach on a large curvature with the formation of 3 folds in the first row of sutures and the second series of continuous suture. After that, a mass of animals and glucose were determined on the 14th, 28th, 42nd and 60th day.Results and Discussion. The level of glucose in the control group on the 60th postoperative day was (18.405 ±1.2) mmol/l, in the first experimental group, where the sleeve resection of the stomach was made, it was (9.539±1.14) mmol/l, in the second group, where the modified gastric plication was made, was (10.39 ±1.49) mmol / l. The determination of glucose in serum on the 60th day after the operation established high statistical significance in decrease of serum glucose between the control and the first experimental group where a sleeve resection was performed (p <0.01) and a high statistical significance in level of serum glucose reduction was observed between the control group and the second experimental group, where a modified stomach plication technique was performed (p <0.01). However, no statistically significant difference between the first and second experimental groups was found (p>0.05).The weight of the body in the control group on the 60th day was (335±17.32) g, in the first experimental group it was on the 60th day (260±12.96) g and in the second experimental group (258.33±11.44) g. The determination of the body weight of rats found a statisti­cally significant decrease in body weight on the 60th postoperative day in rats, which was performed on the sleeve resection of the stomach in comparison with the control group (p <0.01) and a statistically significant reduction in body weight in rats on the 60th post­ operative day after modificated gastric plication in comparison with the control group (p <0.01). However, the statistically significant difference between the weight of the body in rats of the first and second experimental groups on the 60th postoperative day was not revealed (p>0.05).Цель работы: сравнить влияние модифицированной пликации желудка и рукавной резекции желудка у крыс с глутамат- индуцированным ожирением и стрептозотоцин-индуцированным диабетом на динамику снижения массы тела и углеводный обмен.Материалы и методы. Исследование проводили на 45 белых беспородных крысах-самках с исходной массой тела 150–180 г. На животных смоделировано глутамат-индуцированное ожирение и стрептозотоцин-индуцированный диабет 2-го типа. Животные были разделены на 3 группы по 15 животных: контрольную и две экспериментальные, в первой экспериментальной группе было выполнено рукавную резекцию желудка, во второй экспериментальной группе было выполнено пликацию желудка по большой кривизне с формированием 3-х складок в первом ряду швов и с вторым рядом непрерывных швов. После чего измерялась масса животных и глюкоза натощак на 14-е, 28-е, 42-е и 60-е сутки.Результаты исследований и их обсуждение. Уровень глюкозы натощак в контрольной группе на 60-е послеоперационные сутки составил (18,405 ± 1,2) ммоль/л, в первой экспериментальной группе, где было выполнено рукавну резекцию желудка, он составил (9,539 ± 1,14) ммоль/л, во второй группе, где выполнена модифицированная пликация желудка, он составил (10,39 ± 1,49) ммоль/л. Определение глюкозы в сыворотке крови на шестидесятые послеоперационные сутки установило высоко достоверное снижение показателей глюкозы сыворотки крови между контрольной и первой группой, где было выполнено рукавную резекцию (p<0,01) и высоко достоверное снижение глюкозы сыворотки крови между контрольной группой и второй экспериментальной группой, где было выполнено модифицированную методику пликации желудка (p <0.01). Однако статистически значимой разницы между первой и второй экспериментальными группами не выявлено (p>0,05).Масса тела животных контрольной группы на 60-е сутки составляла (335 ± 17,32) г, в первой экспериментальной группе на 60-е сутки она составила (260 ± 12,96) г и во второй экспериментальной группе – (258,33 ± 11,44) г. Определение массы тела крыс установило статистически достоверное снижение массы тела на 60-е послеоперационные сутки у крыс, которым было выполнено рукавную резекцию желудка, по сравнению с контрольной группой (p <0,01) и статистически достоверное снижение массы тела у крыс на 60-е послеоперационные сутки после модифицированной пликации желудка по сравнению с контрольной группой (p <0,01). Однако статистически значимой разницы между массой тела у крыс первой и второй экспериментальной групп на 60-е послеоперационные сутки не обнаружено (p>0,05).Мета роботи: порівняти вплив модифікованої плікації шлунка та рукавної резекції шлунка у щурів із глутамат-індукованим ожирінням та стрептозотоцин-індукованим діабетом на динаміку зниження маси тіла та вуглеводний обмін.Матеріали і методи.  Дослідження проводили на 45 білих безпородних щурах-самках з вихідною масою тіла 150–180 грам. На тваринах було змодельовано глутамат-індуковане ожиріння та стрептозотоцин-індукований діабет 2-го типу. Тварини було розділено на групи по 15 тварин: контрольну та дві експериментальні. В першій експериментальній групі було виконано рукавну резекцію шлунка, другій експериментальній групі було виконано плікацію шлунка по великій кривині з формуванням 3-ох складок і першому ряді швів та другим рядом неприривних швів. Після чого визначали масу тварин та глюкоза натще на 14-ту, 28-му, 42-гу та 60-ту доби.Результати досліджень та їх обговорення. Рівень глюкози натще в контрольній групі на 60-ту післяопераційну добу становив (18,405±1,2) ммоль/л, в першій групі, де було виконано рукавну резекція шлунка, він склав (9,539±1,14) ммоль/л, в другій групі, де виконано модифіковану плікацію шлунка, він склав (10,39±1,49) ммоль/л. Визначення глюкози в сироватці крові на 60-ту – післяопераційну добу встановило високо достовірне зниження показників глюкози сироватки крові між контрольною та першою експериментальною групою, де було виконано рукавну резекцію (p<0,01) та високо достовірне зниження глюкози сироватки крові між контрольною групою та другою експериментальною групою, де було виконано модифіковану методику плікації шлунка (p<0.01). Проте статистичнозначимої різниці між першою та другою експериментальними групами не виявлено (p>0.05). Маса тіла тварин контрольної групи на 60-ту добу становила (335±17,32) г, в першої експериментальної групи на 60-ту добу склала (260±12,96) г та в другій експериментальній групі – (258,33±11,44) г. Визначення маси тіла щурів встановило статистично достовірне зниження маси тіла на 60-ту післяопераційну добу в щурів, яким було виконано рукавну резекцію шлунка, порівняно з контрольною групою (p<0,01) та статистично достовірне зниження маси тіла в щурів на 60-ту післяопераційну добу після модифікованої плікації шлунка порівняно з контрольною групою (p<0,01).  Проте статистично значущої різниці між масою тіла у щурів першої та другої експериментальних груп на 60-ту післяопераційну добу не виявлено (p>0.05)

Cathode fall voltage of TIG arcs from a non-equilibrium arc model

This work presents modelling results concerning a tungsten inert gas (TIG) welding arc. The model provides a consistent description of the free burning arc, the arc attachment and the electrodes. Thermal and chemical non-equilibrium is considered in the whole arc area, and a detailed model of the cathode space-charge sheath is included. The mechanisms in the cathode pre-sheath are treated in the framework of a non-equilibrium approach which is based on a two-fluid description of electrons and heavy particles and a simplified plasma chemistry of argon. A consistent determination of the electrode fall voltages and temperature distributions is achieved. The model is applied to arcs in pure argon at currents up to 250 A, whereby welding of a workpiece made of mild steel with a fixed burner is considered. Arc voltages in the range from 12 to 17 V are obtained at 50 at 250 A, respectively. The space-charge sheath voltage is found to be about 7 V and almost independent of the current. The corresponding temperatures of the cathode tip are in the range from 3,000 K to about 3,800 K. The results obtained are in a good agreement with measurements