Structure-Based Optimization of Selective and Brain Penetrant CK1δ Inhibitors for the Treatment of Circadian Disruptions.

Neuropsychiatric disorders such as major depressive disorders and schizophrenia are often associated with disruptions to the normal 24 h sleep wake cycle. Casein kinase 1 (CK1δ) is an integral part of the molecular machinery that regulates circadian rhythms. Starting from a cluster of bicyclic pyrazoles identified from a virtual screening effort, we utilized structure-based drug design to identify and reinforce a unique "hinge-flip" binding mode that provides a high degree of selectivity for CK1δ versus the kinome. Pharmacokinetics, brain exposure, and target engagement as measured by ex vivo autoradiography are described for advanced analogs.

Mesenchymal stromal cells suppress microglial activation and tumor necrosis factor production.

BACKGROUND AIMS: White matter diseases are commonly associated with microglial activation and neuroinflammation. Mesenchymal stromal cells (MSCs) have immunomodulatory properties and thus have the potential to be developed as cell therapy for white matter disease. MSCs interact with resident macrophages to alter the trajectory of inflammation; however, the impact MSCs have on central nervous system macrophages and the effect this has on the progression of white matter disease are unclear. METHODS: In this study, we utilized numerous assays of varying complexity to model different aspects of white matter disease. These assays ranged from an in vivo spinal cord acute demyelination model to a simple microglial cell line activation assay. Our goal was to investigate the influence of human umbilical cord tissue MSCs on the activation of microglia. RESULTS: MSCs reduced the production of tumor necrosis factor (TNF) by microglia and decreased demyelinated lesions in the spinal cord after acute focal injury. To determine if MSCs could directly suppress the activation of microglia and to develop an efficient potency assay, we utilized isolated primary microglia from mouse brains and the Immortalized MicroGlial Cell Line (IMG). MSCs suppressed the activation of microglia and the release of TNF after stimulation with lipopolysaccharide, a toll-like receptor agonist. CONCLUSIONS: In this study, we demonstrated that MSCs altered the immune response after acute injury in the spinal cord. In numerous assays, MSCs suppressed activation of microglia and release of the pro-inflammatory cytokine TNF. Of these assays, IMG could be standardized and used as an effective potency assay to determine the efficacy of MSCs for treating white matter disease or other neuroinflammatory conditions associated with microglial activation.

Direct Binding Methods to Measure Receptor-Ligand Interactions.

G-protein-coupled receptors (GPCRs) contribute to numerous physiological processes via complex network mechanisms. While indirect signaling assays (Ca2+ mobilization, cAMP production, and GTPγS binding) have been useful in identifying and characterizing downstream signaling mechanisms of GPCRs, these methods lack measurements of direct binding affinities, kinetics, binding specificity, and selectivity that are important parameters in GPCR drug discovery. In comparison to existing direct methods that use radio- or fluorescent labels, label-free techniques can closely emulate the native interactions around binding partners. Surface plasmon resonance (SPR) is a label-free technique that utilizes the refractive index (RI) property and is applied widely in quantitative GPCR-ligand binding kinetics measurement including small molecules screening. However, purified GPCRs are further embedded in a synthetic lipid environment which is immobilized through different tags to the SPR sensor surface, resulting in a non-native environment. Here, we introduced a methodology that also uses the RI property to measure binding interactions in a label-free, immobilization-free arrangement. The free-solution technique is successfully applied in quantifying the interaction of bioactive lipids to cognate lipid GPCRs, which is not purified but rather present in near-native conditions, i.e., in milieu of other cytoplasmic lipids and proteins. To further consider the wide applicability of these free-solution approaches in biomolecular interaction research, additional applications on a variety of receptor-ligand pairs are imperative.

FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis.

Vitamin B12 (B12) deficiency causes neurological manifestations resembling multiple sclerosis (MS); however, a molecular explanation for the similarity is unknown. FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analog approved for MS therapy that can functionally antagonize S1P1. Here, we report that FTY720 suppresses neuroinflammation by functionally and physically regulating the B12 pathways. Genetic and pharmacological S1P1 inhibition upregulates a transcobalamin 2 (TCN2)-B12 receptor, CD320, in immediate-early astrocytes (ieAstrocytes; a c-Fos-activated astrocyte subset that tracks with experimental autoimmune encephalomyelitis [EAE] severity). CD320 is also reduced in MS plaques. Deficiency of CD320 or dietary B12 restriction worsens EAE and eliminates FTY720's efficacy while concomitantly downregulating type I interferon signaling. TCN2 functions as a chaperone for FTY720 and sphingosine, whose complex induces astrocytic CD320 internalization, suggesting a delivery mechanism of FTY720/sphingosine via the TCN2-CD320 pathway. Taken together, the B12-TCN2-CD320 pathway is essential for the mechanism of action of FTY720.

Landscape of In Silico Tools for Modeling Covalent Modification of Proteins: A Review on Computational Covalent Drug Discovery.

Covalent drug discovery has been a challenging research area given the struggle of finding a sweet balance between selectivity and reactivity for these drugs, the lack of which often leads to off-target activities and hence undesirable side effects. However, there has been a resurgence in covalent drug design following the success of several covalent drugs such as boceprevir (2011), ibrutinib (2013), neratinib (2017), dacomitinib (2018), zanubrutinib (2019), and many others. Design of covalent drugs includes many crucial factors, where "evaluation of the binding affinity" and "a detailed mechanistic understanding on covalent inhibition" are at the top of the list. Well-defined experimental techniques are available to elucidate these factors; however, often they are expensive and/or time-consuming and hence not suitable for high throughput screens. Recent developments in in silico methods provide promise in this direction. In this report, we review a set of recent publications that focused on developing and/or implementing novel in silico techniques in "Computational Covalent Drug Discovery (CCDD)". We also discuss the advantages and disadvantages of these approaches along with what improvements are required to make it a great tool in medicinal chemistry in the near future.

Fast and Effective Prediction of the Absolute Binding Free Energies of Covalent Inhibitors of SARS-CoV-2 Main Protease and 20S Proteasome.

The COVID-19 pandemic has been a public health emergency with continuously evolving deadly variants around the globe. Among many preventive and therapeutic strategies, the design of covalent inhibitors targeting the main protease (Mpro) of SARS-CoV-2 that causes COVID-19 has been one of the hotly pursued areas. Currently, about 30% of marketed drugs that target enzymes are covalent inhibitors. Such inhibitors have been shown in recent years to have many advantages that counteract past reservation of their potential off-target activities, which can be minimized by modulation of the electrophilic warhead and simultaneous optimization of nearby noncovalent interactions. This process can be greatly accelerated by exploration of binding affinities using computational models, which are not well-established yet due to the requirement of capturing the chemical nature of covalent bond formation. Here, we present a robust computational method for effective prediction of absolute binding free energies (ABFEs) of covalent inhibitors. This is done by integrating the protein dipoles Langevin dipoles method (in the PDLD/S-LRA/ß version) with quantum mechanical calculations of the energetics of the reaction of the warhead and its amino acid target, in water. This approach evaluates the combined effects of the covalent and noncovalent contributions. The applicability of the method is illustrated by predicting the ABFEs of covalent inhibitors of SARS-CoV-2 Mpro and the 20S proteasome. Our results are found to be reliable in predicting ABFEs for cases where the warheads are significantly different. This computational protocol might be a powerful tool for designing effective covalent inhibitors especially for SARS-CoV-2 Mpro and for targeted protein degradation.

Predicting Mutational Effects on Receptor Binding of the Spike Protein of SARS-CoV-2 Variants.

The pandemic caused by SARS-CoV-2 has cost millions of lives and tremendous social/financial loss. The virus continues to evolve and mutate. In particular, the recently emerged "UK", "South Africa", and Delta variants show higher infectivity and spreading speed. Thus, the relationship between the mutations of certain amino acids and the spreading speed of the virus is a problem of great importance. In this respect, understanding the mutational mechanism is crucial for surveillance and prediction of future mutations as well as antibody/vaccine development. In this work, we used a coarse-grained model (that was used previously in predicting the importance of mutations of N501) to calculate the free energy change of various types of single-site or combined-site mutations. This was done for the UK, South Africa, and Delta mutants. We investigated the underlying mechanisms of the binding affinity changes for mutations at different spike protein domains of SARS-CoV-2 and provided the energy basis for the resistance of the E484 mutant to the antibody m396. Other potential mutation sites were also predicted. Furthermore, the in silico predictions were assessed by functional experiments. The results establish that the faster spreading of recently observed mutants is strongly correlated with the binding-affinity enhancement between virus and human receptor as well as with the reduction of the binding to the m396 antibody. Significantly, the current approach offers a way to predict new variants and to assess the effectiveness of different antibodies toward such variants.

Simulating the directional translocation of a substrate by the AAA+ motor in the 26S proteasome.

This work explored the molecular origin of substrate translocation by the AAA+ motor of the 26S proteasome. This exploration was performed by combining different simulation approaches including calculations of binding free energies, coarse-grained simulations, and considerations of the ATP hydrolysis energy. The simulations were used to construct the free energy landscape for the translocation process. This included the evaluation of the conformational barriers in different translocation steps. Our simulation reveals that the substrate translocation by the AAA+ motor is guided in part by electrostatic interactions. We also validated the experimental observation that bulkier residues in pore loop 1 are responsible for substrate translocation. However, our calculation also reveals that the lysine residues prior to the bulkier residues (conserved along pore loop 1) are also important for the translocation process. We believe that this computational study can help in guiding the ongoing research of the proteasome.

Electrostatically embedded molecules-in-molecules approach and its application to molecular clusters.

We report the application of our fragment-based quantum chemistry model MIM (Molecules-In-Molecules) with electrostatic embedding. The method is termed "EE-MIM (Electrostatically Embedded Molecules-In-Molecules)" and accounts for the missing electrostatic interactions in the subsystems resulting from fragmentation. Point charges placed at the atomic positions are used to represent the interaction of each subsystem with the rest of the molecule with minimal increase in the computational cost. We have carefully calibrated this model on a range of different sizes of clusters containing up to 57 water molecules. The fragmentation methods have been applied with the goal of reproducing the unfragmented total energy at the MP2/6-311G(d,p) level. Comparative analysis has been carried out between MIM and EE-MIM to gauge the impact of electrostatic embedding. Performance of several different parameters such as the type of charge and levels of fragmentation are analyzed for the prediction of absolute energies. The use of background charges in subsystem calculations improves the performance of both one- and two-layer MIM while it is noticeably important in the case of one-layer MIM. Embedded charges for two-layer MIM are obtained from a full system calculation at the low-level. For one-layer MIM, in the absence of a full system calculation, two different types of embedded charges, namely, Geometry dependent (GD) and geometry independent (GI) charges, are used. A self-consistent procedure is employed to obtain GD charges. We have further tested our method on challenging charged systems with stronger intermolecular interactions, namely, protonated ammonia clusters (containing up to 30 ammonia molecules). The observations are similar to water clusters with improved performance using embedded charges. Overall, the performance of NPA charges as embedded charges is found to be the best.

Exploring the Catalytic Reaction of Cysteine Proteases.

Cysteine proteases play a major role in many life processes and are the target of key drugs. The reaction mechanism of these enzymes is a complex process, which involves several steps that are divided into two main groups: acylation and deacylation. In this work, we studied the energy profile for the acylation and a part of the deacylation reaction of three different enzymes, cruzain, papain, and the Q19A-mutated papain with the benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide (CBZ-FR-AMC) substrate. The calculations were performed using the EVB and PDLD/S-LRA methods. The overall agreement between the calculated and observed results is encouraging and indicates that we captured the correct reaction mechanism. Finally, our finding indicates that the minimum of the reaction profile, between the acylation and deacylation steps, should provide an excellent state for the binding of covalent inhibitors.

Exploring the Proteolysis Mechanism of the Proteasomes.

The proteasome is a key protease in the eukaryotic cells which is responsible for various important cellular processes such as the control of the cell cycle, immune responses, protein homeostasis, inflammation, apoptosis, and the response to proteotoxic stress. Acting as a major molecular machine for protein degradation, proteasome first identifies damaged or obsolete regulatory proteins by attaching ubiquitin chains and subsequently utilizes conserved pore loops of the heterohexameric ring of AAA+ (ATPases associated with diverse cellular activities) to pull and mechanically unfold and translocate the misfolded protein to the active site for proteolysis. A detailed knowledge of the reaction mechanism for this proteasomal proteolysis is of central importance, both for fundamental understanding and for drug discovery. The present study investigates the mechanism of the proteolysis by the proteasome with full consideration of the protein's flexibility and its impact on the reaction free energy. Major attention is paid to the role of the protein electrostatics in determining the activation barriers. The reaction mechanism is studied by considering a small artificial fluorogenic peptide substrate (Suc-LLVY-AMC) and evaluating the activation barriers and reaction free energies for the acylation and deacylation steps, by using the empirical valence bond method. Our results shed light on the proteolysis mechanism and thus should be important for further studies of the proteasome action.

Cln1-mutations suppress Rab7-RILP interaction and impair autophagy contributing to neuropathology in a mouse model of infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease (LSD) caused by inactivating mutations in the CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme that catalyzes the deacylation of S-palmitoylated proteins to facilitate their degradation and clearance by lysosomal hydrolases. Despite the discovery more than two decades ago that CLN1 mutations causing PPT1-deficiency underlies INCL, the precise molecular mechanism(s) of pathogenesis has remained elusive. Here, we report that autophagy is dysregulated in Cln1-/- mice, which mimic INCL and in postmortem brain tissues as well as cultured fibroblasts from INCL patients. Moreover, Rab7, a small GTPase, critical for autophagosome-lysosome fusion, requires S-palmitoylation for trafficking to the late endosomal/lysosomal membrane where it interacts with Rab-interacting lysosomal protein (RILP), essential for autophagosome-lysosome fusion. Notably, PPT1-deficiency in Cln1-/- mice, dysregulated Rab7-RILP interaction and preventing autophagosome-lysosome fusion, which impaired degradative functions of the autolysosome leading to INCL pathogenesis. Importantly, treatment of Cln1-/- mice with a brain-penetrant, PPT1-mimetic, small molecule, N-tert (butyl)hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal a previously unrecognized role of CLN1/PPT1 in autophagy and suggest that small molecules functionally mimicking PPT1 may have therapeutic implications.

Health-Care Waste Management in Public Sector of Tripura, North-East India: An Observational Study.

BACKGROUND: Hospitals generate variety of waste which is hazardous to patients, health workers, community, and environment. Proper health-care waste management (HCWM) requires infrastructure, trained workforce, law and supervision. More than 80% of the population of Tripura depends on the public health-care system but the knowledge and practice of health-care workers regarding HCWM in the public sector of Tripura is not clear. OBJECTIVES: The objective was to assess the knowledge and practice of health-care workers regarding HCWM and to take an account of the existing HCWM facilities in the public sector of Tripura. STUDY DESIGN: This was a facility-based, cross-sectional study. MATERIALS AND METHODS: This study was conducted during 1st November 2015 to 16th October 2017 among 544 health-care workers working in thirty health institutions chosen by stratified random sampling. Data entry and analysis was performed using SPSS software version 15.0. RESULTS: Overall, 37.68% of the respondents had fair knowledge regarding HCWM, 8.27% received in-service training on HCWM, 66.17% were immunized against hepatitis B and > 90% of the respondents knew about segregation of waste at source but knowledge regarding the use of colored bins for this purpose varied widely across different categories of participants. Housekeeping staff were ignorant about most of these issues. The importance of disinfecting the waste before disposal was known to 83.63% of the workers. Proper HCWM was practiced by 39.15% and segregation of waste at source into colored bins was followed by 23.3% of the respondents. The study revealed both waste management facilities and display of waste management policy as poor. Technical qualification and in-service training were identified as the statistically significant determinants of knowledge and practice of HCWM (P < 0.05). CONCLUSION: HCWM scenario including knowledge of health-care workers in Tripura is lacking. Installing proper waste management facilities, raising technical qualification at recruitment and in-service training may improve the situation.

Human umbilical cord blood monocytes, but not adult blood monocytes, rescue brain cells from hypoxic-ischemic injury: Mechanistic and therapeutic implications.

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.

Predicting the Binding of Fatty Acid Amide Hydrolase Inhibitors by Free Energy Perturbation.

Since a goal of most drug discovery projects in either academia or industry is to design molecules that selectively bind to the desired protein, determination of protein-ligand binding free energies is of utmost importance in computer aided drug design. With the help of significant improvements in computer power, enhanced sampling techniques and accuracy of force fields, FEP (free energy perturbation) is becoming an important tool to estimate binding free energies in many drug discovery projects both retrospectively and prospectively. We have evaluated the ability of Schrödinger's FEP+ to predict relative binding free energies of a congeneric series of noncovalent fatty acid amide hydrolase (FAAH) inhibitors using an in-house crystal structure. This study shows that although an impressively accurate correlation can be obtained with experimental IC50s considering small perturbations on the deeper side of the pocket, the same was not observed with small perturbations on the relatively more open-ended and solvent-accessible side of the pocket. To understand these observations, we thoroughly investigated several key factors including the sampling of asymmetrically substituted rings, different perturbation maps, impact of simultaneous perturbations at two different ends of the ligand, and selecting the perturbations in a "chemically sensible" way.

An Analysis of Different Components of a High-Throughput Screening Library.

Since many projects at pharmaceutical organizations get their start from a high-throughput screening (HTS) campaign, improving the quality of the HTS deck can improve the likelihood of discovering a high-quality lead molecule that can be progressed to a drug candidate. Over the past decade, Janssen has implemented several strategies for external compound acquisition to augment the screening deck beyond the chemical space and number of molecules synthesized for internal projects. In this report, we analyzed the performance of each of those compound collections in the screening campaigns performed internally within Janssen during the last five years. We classified the screening library into two broad categories: Internal and External. The comparison of the performance of these sets of libraries was done by considering the primary, confirmation, and dose response hit rates. Our analysis revealed that Internal compounds (resulting from numerous medicinal chemistry efforts against diverse protein targets) have higher average confirmation hit rates than External ones; however, actives from both categories show similar probabilities of hitting multiple distinct targets. We also investigated the property landscape of both sets of libraries to identify the key elements which make a difference in these categories of compounds. From this analysis, Janssen aims to understand the descriptor landscape of the compounds with the highest hit rates and to use them for improving its future acquisition strategies as well as to inform our plating strategy.

Gene products promoting remyelination are up-regulated in a cell therapy product manufactured from banked human cord blood.

BACKGROUND AIMS: DUOC-01, a cell product being developed to treat demyelinating conditions, is composed of macrophages that arise from CD14+ monocytes in the mononuclear cell (MNC) population of banked cord blood (CB). This article demonstrates that expression of multiple gene products that promote remyelination is rapidly up-regulated during manufacturing of DUOC-01 from either MNC or purified CB CD14+ monocytes. METHODS: Cell cultures were initiated with MNC or with immunoselected CD14+ monocytes isolated from the same CB unit. Cell products present in these cultures after 2 and 3 weeks were compared by three methods. First, quantitative polymerase chain reaction was used to compare expression of 77 transcripts previously shown to be differentially expressed by freshly isolated, uncultured CB CD14+ monocytes and DUOC-01. Second, accumulation of 16 soluble proteins in the culture medium was measured by Bioplex methods. Third, whole transcriptomes of the cell products were compared by microarray analysis. RESULTS: Key transcripts in multiple pathways that promote remyelination were up-regulated in DUOC-01, and substantial secretion of proteins corresponding to many of these transcripts was detected. Cell products manufactured from MNC or from CD14+ monocytes were similar with regard to all metrics. Upregulation of gene products characteristic of DUOC-01 was largely completed within 14 days of culture. CONCLUSION: We demonstrate that expression of multiple gene products that promote remyelination is up-regulated during the first 2 weeks of manufacturing of DUOC-01. Measuring these mechanistically important transcripts and proteins will be useful in monitoring manufacturing, evaluating manufacturing changes, and developing mechanism-based product potency assays.

Bond Activation and Hydrogen Evolution from Water through Reactions with M3S4 (M = Mo, W) and W3S3 Anionic Clusters.

Transition metal sulfides (TMS) are being investigated with increased frequency because of their ability to efficiently catalyze the hydrogen evolution reaction. We have studied the trimetallic TMS cluster ions, Mo3S4-, W3S4-, and W3S3-, and probed their efficiency for bond activation and hydrogen evolution from water. These clusters have geometries that are related to the edge sites on bulk MoS2 surfaces that are known to play a role in hydrogen evolution. Using density functional theory, the electronic structures of these clusters and their chemical reactivity with water have been investigated. The reaction mechanism involves the initial formation of hydroxyl and thiol groups, hydrogen migration to form an intermediate with a metal hydride bond, and finally, combination of a hydride and a proton to eliminate H2. Using this mechanism, free energy profiles of the reactions of the three metal clusters with water have been constructed. Unlike previous reactivity studies of other related cluster systems, there is no overall energy barrier in the reactions involving the M3S4 systems. The energy required for the rate-determining step of the reaction (the initial addition of the cluster by water) is lower than the separated reactants (-0.8 kcal/mol for Mo and -5.1 kcal/mol for W). They confirm the M3S4- cluster's ability to efficiently activate the chemical bonds in water to release H2. Though the W3S3- cluster is not as efficient at bond activation, it provides insights into the factors that contribute to the success of the M3S4 anionic systems in hydrogen evolution.

Hydrogen evolution from water using Mo-oxide clusters in the gas phase: DFT modeling of a complete catalytic cycle using a Mo2O4-/Mo2O5- cluster couple.

Density functional theory (DFT) calculations using a small metal cluster couple, Mo2O4-/Mo2O5-, are used to model a complete catalytic cycle for H2 production from water. While Mo2O4- is known to readily react with water to form Mo2O5- and release H2, the principal challenge is in reducing Mo2O5- to Mo2O4- to complete the cycle. We investigate the role of several potential sacrificial reagents (ethylene, propylene, CO and acetylene) that can reduce Mo2O5- after the initial oxidation. DFT calculations of the free energy reaction pathways demonstrate the presence of overall kinetically accessible barriers that are below the entrance channel (separated reactants) in the Mo2O4- + H2O reaction (step I) followed by the Mo2O5- + sacrificial reagent reactions (step II). Though the overall reaction is thermodynamically favorable, the first step is highly exothermic while the second step is endothermic. The deepest part of the potential energy surface is a complex of Mo2O5- with the sacrificial reagent. If the energy gained in the first reaction and the succeeding complex formation is not lost due to collisions, the subsequent barriers can be overcome, leading to possible catalytic applications of the Mo2O4-/Mo2O5- cluster couple in H2 production reactions.