The Role of Structural Biology Task Force: Validation of the Binding Mode of Repurposed Drugs Against SARS-CoV-2 Protein Targets: Focus on SARS-CoV-2 Main Protease (Mpro): A Promising Target for COVID-19 Treatment

The main protease (Mpro) of SARS-CoV-2, a cysteine protease that plays a key role in generating the active proteins essential for coronavirus replication, is a validated drug target for treating COVID-19. The structure of Mpro has been elucidated by macromolecular crystallography, but owing to its conformational flexibility, finding effective inhibitory ligands was challenging. Screening libraries of ligands as part of EXaSCale smArt pLatform Against paThogEns (ExScalate4CoV) yielded several potential drug molecules that inhibit SARS-CoV-2 replication in vitro. We solved the crystal structures of Mpro in complex with repurposed drugs like myricetin, a natural flavonoid, and MG-132, a synthetic peptide aldehyde. We found that both inhibitors covalently bind the catalytic cysteine. Notably, myricetin has an unexpected binding mode, showing an inverted orientation with respect to that of the flavonoid baicalein. Moreover, the crystallographic model validates the docking pose suggested by molecular dynamics experiments. The mechanism of MG-132 activity against SARS-CoV-2 Mpro was elucidated by comparison of apo and inhibitor-bound crystals, showing that regardless of the redox state of the environment and the crystalline symmetry, this inhibitor binds covalently to Cys145 with a well-preserved binding pose that extends along the whole substrate binding site. MG-132 also fits well into the catalytic pocket of human cathepsin L, as shown by computational docking, suggesting that it might represent a good start to developing dual-targeting drugs against COVID-1

Known drugs identified by structure-based virtual screening are able to bind sigma-1 receptor and increase growth of huntington disease patient-derived cells

Huntington disease (HD) is a devastating and presently untreatable neurodegenerative disease characterized by progressively disabling motor and mental manifestations. The sigma-1 receptor (σ1R) is a protein expressed in the central nervous system, whose 3D structure has been recently determined by X-ray crystallography and whose agonists have been shown to have neuro-protective activity in neurodegenerative diseases. To identify therapeutic agents against HD, we have implemented a drug repositioning strategy consisting of: (i) Prediction of the ability of the FDA-approved drugs publicly available through the ZINC database to interact with σ1R by virtual screening, followed by computational docking and visual examination of the 20 highest scoring drugs; and (ii) Assessment of the ability of the six drugs selected by computational analyses to directly bind purified σ1R in vitro by Surface Plasmon Resonance and improve the growth of fibro-blasts obtained from HD patients, which is significantly impaired with respect to control cells. All six of the selected drugs proved able to directly bind purified σ1R in vitro and improve the growth of HD cells from both or one HD patient. These results support the validity of the drug repositioning procedure implemented herein for the identification of new therapeutic tools against HD

Cavity and Cryomodule Developments for EIC

The EIC is a major new project under construction at BNL in partnership with JLab. It relies upon a number of new SRF cavities at 197 MHz, 394 MHz, 591 MHz and 1773 MHz to pre-bunch, accelerate, cool and crab the stored beams. R&D is focusing on the 591 MHz elliptical cavity and 197 MHz crab cavity first as these are the most challenging. Preliminary designs of these cavities are presented along with an R&D status report. To avoid developing multiple different cryostats a modular approach is adopted using a high degree of commonality of parts and systems. This approach may be easily adapted to other frequencies and applications

Linking Inflammation to Natural Killer T Cell Activation

Immune activation is often associated with inflammation, but inflammation's role in the expansion of antigen-specific immune responses remains unclear. This primer focuses on recent findings that show how specific natural killer T cells are activated by inflammatory messengers, thus illuminating the cellular and molecular links between immunity and inflammation

Determination of Cellular Lipids Bound to Human CD1d Molecules

CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death

Tailored design of NKT-stimulatory glycolipids for polarization of immune responses

Natural killer T (NKT) cell is a distinct population of T lymphocytes that can rapidly release massive amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipids presented by CD1d. The secreted cytokines can promote cell-mediated immunity to kill tumor cells and intracellular pathogens, or suppress autoreactive immune cells in autoimmune diseases. Thus, NKT cell is an attractive target for developing new therapeutics to manipulate immune system. The best-known glycolipid to activate NKT cells is α-galactosylceramide (α-GalCer), which has been used as a prototype for designing new NKT stimulatory glycolipids. Many analogues have been generated by modification of the galactosyl moiety, the acyl chain or the phytosphingosine chain of α-GalCer. Some of the analogues showed greater abilities than α-GalCer in polarizing immune responses toward Th1 or Th2 dominance. Among them, several analogues containing phenyl groups in the lipid tails were more potent in inducing Th1-skewed cytokines and exhibited greater anticancer efficacy than α-GalCer. Analyses of the correlation between structure and activity of various α-GalCer analogues on the activation of iNKT cell revealed that CD1d–glycolipid complexes interacted with the same population of iNKT cell expressing similar T-cell receptor Vβ as α-GalCer. On the other hand, those phenyl glycolipids with propensity for Th1 dominant responses showed greater binding avidity and stability than α-GalCer for iNKT T-cell receptor when complexed with CD1d. Thus, it is the avidity and stability of the ternary complexes of CD1d-glycolipid-iNKT TCR that dictate the polarity and potency of immune responses. These findings provide a key to the rationale design of immune modulating glycolipids with desirable Th1/Th2 polarity for clinical application. In addition, elucidation of α-GalCer-induced anergy, liver damage and accumulation of myeloid derived suppressor cells has offered explanation for its lacklustre anti-cancer activities in clinical trials. On other hand, the lack of such drawbacks in glycolipid analogues containing phenyl groups in the lipid tails of α-GalCer coupled with the greater binding avidity and stability of CD1d-glycolipid complex for iNKT T-cell receptor, account for their superior anti-cancer efficacy in tumor bearing mice. Further clinical development of these phenyl glycolipids is warranted

Combination of SAXS and protein painting discloses the three-dimensional organization of the bacterial cysteine synthase complex, a potential target for enhancers of antibiotic action

The formation of multienzymatic complexes allows for the fine tuning of many aspects of enzymatic functions, such as efficiency, localization, stability, and moonlighting. Here, we investigated, in solution, the structure of bacterial cysteine synthase (CS) complex. CS is formed by serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase isozyme A (CysK), the enzymes that catalyze the last two steps of cysteine biosynthesis in bacteria. CysK and CysE have been proposed as potential targets for antibiotics, since cysteine and related metabolites are intimately linked to protection of bacterial cells against redox damage and to antibiotic resistance. We applied a combined approach of small-angle X-ray scattering (SAXS) spectroscopy and protein painting to obtain a model for the solution structure of CS. Protein painting allowed the identification of protein-protein interaction hotspots that were then used as constrains to model the CS quaternary assembly inside the SAXS envelope. We demonstrate that the active site entrance of CysK is involved in complex formation, as suggested by site-directed mutagenesis and functional studies. Furthermore, complex formation involves a conformational change in one CysK subunit that is likely transmitted through the dimer interface to the other subunit, with a regulatory effect. Finally, SAXS data indicate that only one active site of CysK is involved in direct interaction with CysE and unambiguously unveil the quaternary arrangement of CS

Investigating Drug-Target Residence Time in Kinases through Enhanced Sampling Simulations

It is widely accepted that drug-target association and dissociation rates directly affect drug efficacy and safety. To rationally optimize drug binding kinetics, one must know the atomic arrangement of the protein-ligand complex during the binding/unbinding process in order to detect stable and metastable states. Whereas experimental approaches can determine kinetic constants with fairly good accuracy, computational approaches based on molecular dynamics (MD) simulations can deliver the atomistic details of the unbinding process. Furthermore, they can also be utilized prospectively to predict residence time (i.e., the inverse of unbinding kinetics constant, koff) with an acceptable level of accuracy. Here, we report a novel method based on adiabatic bias MD with an electrostatics-like collective variable (dubbed elABMD) for sampling protein-ligand dissociation events in two kinases. elABMD correctly ranked a ligand series on glucokinase, in agreement with experimental data and previous calculations. Subsequently, we applied the new method prospectively to a congeneric series of GSK-3\u3b2 inhibitors. For this series, new crystal structures were generated and the residence time was experimentally measured with surface plasmon resonance (SPR). There was good agreement between computational predictions and experimental measures, suggesting that elABMD is an innovative and efficient tool for calculating residence times

Polymutant BCR-ABL1 proteins during chronic myeloid leukemia therapy: novel mechanisms of resistance from clinical, in vitro, and in silico evidences

The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated with imatinib failure among patients with chronic myeloid leukemia. Typically, patients failing imatinib undergo sequential therapy with second line tyrosine kinase inhibitors (TKIs) such as nilotinib or dasatinib. To investigate the prevalence of BCR-ABL1 mutations and the mechanisms of acquired TKI resistance during sequential therapy, we utilized a sensitive cloning technique in a cohort of chronic-phase patients failing imatinib therapy that subsequently received dasatinib. The analysis revealed that most patients failing imatinib carry BCR-ABL1 mutations, many with polymutant BCR-ABL1 alleles, and that there is progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of treatment. In silico modeling of the most frequent polymutants demonstrated profound resistance to imatinib and second line TKIs. Modeling of BCR-ABL1 in complex with the recently approved TKI ponatinib highlighted potentially effective therapeutic strategies for patients carrying these recalcitrant and complex BCR-ABL1 mutant proteins while unveiling novel mechanisms of escape to ponatinib therapy