TREADS: Target Research for Anti-epileptic Drugs Using Data Science.

CONTEXT: Epilepsy is a common neurological disease and is classified into different types based on features such as the kind of seizure, age of onset, part of brain effected, etc. There are nearly 30 approved anti-epileptic drugs (AEDs) for treating different epilepsies and each drug targets proteins exhibiting a specific molecular mechanism of action. There are many genes, proteins, and microRNAs known to be associated with different epileptic disorders. This rich information on epilepsy-associated data is not available at one single location and is scattered across multiple publicly available repositories. There is a need to have a single platform integrated with the data, as well as tools required for epilepsy research. METHODS AND MATERIAL: Text mining approaches are used to extract data from multiple biological sources. The data is curated and populated within an in-house developed epilepsy database. Machine-learning based models are built in-house to know the probability of a protein being druggable based on the significant protein features. A web interface is provided for the access of the epilepsy database as well as the ML-based tool developed in-house. RESULTS: The epilepsy-associated data is made accessible through a web browser. For a protein of interest, the platform provides all the feature values, and the results generated using different machine learning models are displayed as visualization plots. CONCLUSIONS: To meet these objectives, we present TREADS, a platform for epilepsy research community, having both database and an ML-based tool for the study of AED targets. TO ACCESS TREADS: https://treads-aer.cdacb.in.

Molecular modelling reveals how abundance of α4 sub-type in synaptic GABARA receptor can lead to refractoriness toward GABA and BZ-type drugs.

Epilepsy is a complex neurological disorder with genetic and acquired causes, and the drugs presently used to treat epilepsy are not effective in about 30% of the cases. Identification of the molecular mechanisms of resistance will help in the development of newer molecules for treatment. Recent clinical data indicate increased expression of α4- and γ2-containing synaptic GABARA receptors in patients of focal cortical dysplasia (FCD), which is associated with refractory epilepsy pathology. We have investigated, by molecular modelling and docking, the structure and ligand-binding efficiency of the α4-containing hetero-pentameric synaptic GABARA receptor. Though the overall conformation is similar to that of the α1-containing receptor, local conformational changes are seen due to differences between aligned α1 and α4 sub-type residues. The overlaps ALA209(α1)/PRO215(α4) and PHE73(α1)/TYR79(α4) have together caused conformational changes in ARG100(α4) (aligned with ARG94 in α1) thereby affecting key hydrogen bonding interactions with the inhibitory neurotransmitter GABA. This may influence the nature of seizures as strength of GABA-binding is known to affect the nature of Inhibitory Post-Synaptic Currents (IPSCs) from GABAergic neurons. The residue ARG135 (α4) aligns with the residue HIS129 (α1) in the benzodiazapine binding pocket. Molecular modelling also shows that a steric clash between benzodiazapine-type (BZ-type) drugs and ARG135 would reduce the binding of BZ-type drugs to α4-containing receptor. These two findings rationalize the observed association between over-expression of α4-containing synaptic GABARA receptors and refractory epilepsy pathology in FCD. The accurate three-dimensional geometry of the receptor-drug complex made available by these modelling studies will help in designing effective drugs.Communicated by Ramaswamy H. Sarma.

A computational study on hydroxychloroquine binding to target proteins related to SARS-COV-2 infection.

COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has posed a global health emergency. Repurposing of existing drugs can be a rapid and effective strategy to fight the infection. Clinical trials have reported reduction or elimination of viral load when patients were treated with the anti-malarial drug Hydroxychloroquine (HCQ). To understand the molecular mechanism of action for effective repurposing of this drug we have carried out in silico docking and dynamics studies on complexes between HCQ and target proteins, which were identified through both literature survey and structural similarity searches in databases of small molecule - protein complexes. The proteins identified as binding HCQ are: Angiotensin Converting Enzyme 2 (ACE2), α7 nicotinic AcetylCholine Receptor (α7 nAChR), α1D-adrenergic receptor (α1D-AR), Histamine N- Methyl Transferase (HNMT) and DNA gyrase/Topoisomerase III ß (Top3ß). The majority of these proteins are novel and have not been used before, in docking studies. Our docking and simulation results support action of HCQ both at the entry and post-entry stages of SARS-CoV2 infection. The mechanism of action at the entry stage is through blocking the virus-binding sites on the two receptors, ACE2 & α7 nAChR, by binding directly at those sites. Our computational studies also show that the action of HCQ at the post-entry stage is to prevent both viral replication and generation of 'cytokine storm' by inhibiting host Top3ß enzyme and α1D-AR, respectively. Binding of HCQ to HNMT is not a desired binding, and therefore this should be reduced during repurposing of HCQ.

X-ray crystallographic analysis of time-dependent binding of guanidine hydrochloride to HEWL: First steps during protein unfolding.

Time-dependent binding of guanidine hydrochloride (GuHCl) to hen egg-white lysozyme (HEWL), and effects of this binding on the protein structure have been investigated by solving X-ray structures of crystalline complexes. The complexes have been prepared by soaking, for different periods of time, native lysozyme crystals in solutions containing 2.5M GuHCl. In the refined structures, the number of water molecules in the protein's first solvent shell has progressively decreased from 152 to 115, showing protein's preference for guanidinium over water. Guanidinium ions preferentially hydrogen bond with the backbone carbonyl oxygen atoms. In their van der Waals interactions, they do not show any preference for apolar residues. Guanidinium ions have replaced water molecules that form cages around exposed hydrophobic residues. Guanidinium binding has decreased the average length of water-water hydrogen bond by 0.1Å. The hydrogen bonds between main chain atoms have been weakened by GuHCl, and this may be the reason for increased potency of GuHCl compared to urea. Guanidinium binding destabilizes the ß-domain by causing loss of hydrogen bonds involving Asn 59 side chain. Interestingly, this loss is almost identical to that observed in structures of amyloidogenic variants of human lysozyme. Compounds preventing this loss could be anti-amyloidogenic.

Structural basis to characterise transactivation domain of BRCA1.

Familial inheritance of breast and ovarian cancer is attributed to mutations discovered in functional domains of BRCA1 gene. BRCA1 is a multifunctional protein responsible for maintaining the genomic integrity and has transcriptional regulatory function encoded in its C-terminal region. The different amino-terminal e extensions to BRCA1 BRCT domain are responsible for transcription activation. However, only BRCA1 BRCT (1649-1859) amino acids have been explored for its structural characteristics. Noting the importance of extended region to the N-terminus of BRCT different regions of BRCA1 which demonstrates maximum transactivation activity has been explored for their structure and functional activity. Secondary and tertiary structural analysis revealed a limited alpha-helical content with well-folded tertiary structure. In silico tools were used to corroborate the in vitro results. Amino acids composition and sequence analysis display a propensity for intrinsic disorder and coiled-coil formation in BRCA1 (1396-1863) (BRCA1-TAD). The results presented in this paper suggest the extreme flexibility in coiled-coil motif might be an important requirement in the establishment of protein-protein interaction networks for BRCA1.

Functional Basis and Biophysical Approaches to Characterize the C-Terminal Domain of Human-Ribosomal S6 Kinases-3.

Ribosomal S6 kinases (RSKs) are the major functional components in mitogen-activated protein kinase (MAPK) pathway, and these are activated by upstream Extracellular signal-regulated kinase. Upon activation, RSKs activate a number of substrate molecules involved in transcription, translation and cell-cycle regulation. But how cellular binding partners are engaged in the MAPK pathways and regulate the molecular mechanisms have not been explored. Considering the importance of protein-protein interactions in cell signalling and folding pattern of native protein, functional C-terminal kinase domain of RSK3 has been characterized using in vitro, in silico and biophysical approaches. RSKs discharge different functions by binding to downstream kinase partners. Hence, depending upon cellular binding partners, RSKs translocate between cytoplasm and nucleus. In our study, it has been observed that the refolded C-terminal Kinase domain (CTKD) of RSK 3 has a compact domain structure which is predominantly α-helical in nature by burying the tryptophans deep into the core, which was confirmed by CD, Fluorescence spectroscopy and limited proteolysis assay. Our study also revealed that RSK 3 CTKD was found to be a homotrimer from DLS experiments. A model was also built for RSK 3 CTKD and was further validated using PROCHECK and ProSA webservers.

Protein crystallography beamline (PX-BL21) at Indus-2 synchrotron.

The protein crystallography beamline (PX-BL21), installed at the 1.5 T bending-magnet port at the Indian synchrotron (Indus-2), is now available to users. The beamline can be used for X-ray diffraction measurements on a single crystal of macromolecules such as proteins, nucleic acids and their complexes. PX-BL21 has a working energy range of 5-20 keV for accessing the absorption edges of heavy elements commonly used for phasing. A double-crystal monochromator [Si(111) and Si(220)] and a pair of rhodium-coated X-ray mirrors are used for beam monochromatization and manipulation, respectively. This beamline is equipped with a single-axis goniometer, Rayonix MX225 CCD detector, fluorescence detector, cryogenic sample cooler and automated sample changer. Additional user facilities include a workstation for on-site data processing and a biochemistry laboratory for sample preparation. In this article the beamline, other facilities and some recent scientific results are briefly described.

Multimodal approach to explore the pathogenicity of BARD1, ARG 658 CYS, and ILE 738 VAL mutants.

BARD1-BRCA1 complex plays an important role in DNA damage repair, apoptosis, chromatin remodeling, and other important processes required for cell survival. BRCA1 and BARD1 heterodimer possess E3 ligase activity and is involved in genome maintenance, by functioning in surveillance for DNA damage, thereby regulating multiple pathways including tumor suppression. BRCT domains are evolutionary conserved domains present in different proteins such as BRCA1, BARD1, XRCC, and MDC1 regulating damage response and cell-cycle control through protein-protein interactions. Nonetheless, the role of BARD1BRCT in the recruitment of DNA repair mechanism and structural integrity with BRCA1 complex is still implicit. To explicate the role of BARD1BRCT in the DNA repair mechanism, in silico, in vitro, and biophysical approach were applied to characterize BARD1 BRCT wild-type and Arg658Cys and Ile738Val mutants. However, no drastic secondary and tertiary structural changes in the mutant proteins were observed. Thermal and chemical denaturation studies revealed that mutants Arg658Cys and Ile738Val have a decrease in Tm and ∆G than the wild type. In silico studies of BARD1 BRCT (568-777) and mutant protein indicate loss in structural compactness on the Ile738Val mutant. Comparative studies of wild-type and mutants will thus be helpful in understanding the basic role of BARD1BRCT in DNA damage repair.

Functional assessment of intrinsic disorder central domains of BRCA1.

The most studied function of BRCA1 is that of tumor suppression through its role in DNA repair and transcription regulation. Germline mutations discovered in a larger cohort of patients, abrogate BRCA1 interactions with reported cellular partners, and are responsible for breast and ovarian cancer. The different functional regions of BRCA1 interact with nearly 30 different cellular partners. Thus, it becomes clinically significant to understand the detailed protein-protein interactions associated with functional regions of BRCA1. Different overlapping central domains of BRCA1 have been characterized using in silico, in vitro and biophysical approaches. To our conclusions, it has been observed that central domains of BRCA1 are intrinsically disordered and has large hydrodynamic radius with random coil like structures.

Tetrameric ZBRK1 DNA binding domain has affinity towards cognate DNA in absence of zinc ions.

Zinc finger transcription regulatory proteins play crucial roles in cell-cycle regulation, DNA damage response and tumor genesis. Human ZBRK1 is a zinc-finger transcription repressor protein, which recognizes double helical DNA containing consensus sequences of 5'GGGXXXCAGXXXTTT3'. In the present study, we have purified recombinant DNA binding domain of ZBRK1, and studied binding with zinc ions and DNA, using biophysical techniques. The elution profile of the purified protein suggests that this ZBRK1 forms a homotetramer in solution. Dissociation and pull down assays also suggest that this domain forms a higher order oligomer. The ZBRK1-DNA binding domain acquires higher stability in the presence of zinc ions and DNA. The secondary structure of the ZBRK1-DNA complex is found to be significantly altered from the standard B-DNA conformation.

Preliminary crystallographic studies of BRCA1 BRCT-ABRAXAS complex.

The BRCA1 holoenzyme complex plays an important role in DNA damage repair. ABRAXAS is a newly discovered component of this complex and its C-terminal region directly binds to the BRCA1 BRCT domain. Single crystals of the BRCA1 BRCT-ABRAXAS complex grown by co-crystallization belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 187.18, c = 85.31 Å. Diffraction data were collected on the BM-14 beamline at the ESRF. Molecular-replacement calculations using Phaser led to three molecules in the asymmetric unit and a high solvent content of 76%.

Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex.

The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

X-ray snapshot of HIV-1 protease in action: observation of tetrahedral intermediate and short ionic hydrogen bond SIHB with catalytic aspartate.

Structural snapshots of each step in the catalytic cycle would help development of inhibitors of human immunodeficiency virus type 1 protease (HIV-1 PR) as effective drugs against HIV/AIDS. We report here one snapshot obtained by determining the structure of enzyme-substrate complex under conditions where the catalytic activity of the enzyme is greatly reduced. The 1.76 A crystal structure shows the oligopeptide substrate, AETFYVDGAA, converted in situ into a gem-diol tetrahedral intermediate (TI). The gem-diol intermediate is neutral and one of the hydroxyl oxygens forms a very short hydrogen bond (2.2 A) with the anionic aspartate of the catalytic dyad, which is monoprotonated. Further, there is no hydrogen atom on the outer oxygen of the neutral aspartate. These two observations provide direct evidence that, in the reaction mechanism, hydrogen bonding between catalytic aspartate and scissile carbonyl oxygen facilitates water attack on the scissile carbon atom. Comparison with the structural snapshot of the biproduct complex involving the same substrate reveals the reorganization of the hydrogen bonds at the catalytic center as the enzymatic reaction progresses toward completion. Accumulation of TI in the crystals provides direct evidence that collapse of TI is the rate-limiting step of hydrolysis.

Resistance mechanism revealed by crystal structures of unliganded nelfinavir-resistant HIV-1 protease non-active site mutants N88D and N88S.

Nelfinavir is an inhibitor of HIV-1 protease, and is used for treatment of patients suffering from HIV/AIDS. However, treatment results in drug resistant mutations in HIV-1 protease. N88D and N88S are two such mutations which occur in the non-active site region of the enzyme. We have determined crystal structures of unliganded N88D and N88S mutants of HIV-1 protease to resolution of 1.65A and 1.8A, respectively. These structures refined against synchrotron data lead to R-factors of 0.1859 and 0.1780, respectively. While structural effects of N88D are very subtle, the mutation N88S has caused a significant conformational change in D30, an active site residue crucial for substrate and inhibitor binding.

Crystallization and preliminary X-ray crystallographic analysis of PhoK, an extracellular alkaline phosphatase from Sphingomonas sp. BSAR-1.

Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 87.37, c = 168.16 A, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95 A resolution at the ESRF.

X-ray structure of HIV-1 protease in situ product complex.

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.

Conformation and structure of bis(glycyl-L-aspartic acid) oxalate 0.4-hydrate.

The title bis(glycyl-L-aspartic acid) oxalate complex {systematic name: bis[2-(2-ammonioacetamido)butanedioic acid] oxalate 0.4-hydrate}, 2C6H11N2O5+.C2O4(2-).4H2O, crystallizes in a triclinic space group with the planar peptide unit in a trans conformation. The asymmetric unit consists of two glycyl-L-aspartic acid molecules with positively charged amino groups and neutral carboxyl groups, and an oxalate dianion. The twist around the C-Calpha bond indicates that both the peptide molecules adopt extended conformations, while the twist around the N-Calpha bond shows that one has a folded and the other a semi-extended state. The present complex can be described as an inclusion compound with the dipeptide molecule as the host and the oxalate anion as the guest. The usual head-to-tail sequence of aggregation is not observed in this complex, as is also the case with the glycyl-L-aspartic acid dihydrate molecule. The study of aggregation and interaction patterns in binary systems is the first step towards understanding more complex phenomena. This further leads to results that are of general interest in bimolecular aggregation.

Crystal structure of HIV-1 protease in situ product complex and observation of a low-barrier hydrogen bond between catalytic aspartates.

HIV-1 protease is an effective target for designing drugs against AIDS, and structural information about the true transition state and the correct mechanism can provide important inputs. We present here the three-dimensional structure of a bi-product complex between HIV-1 protease and the two cleavage product peptides AETF and YVDGAA. The structure, refined against synchrotron data to 1.65 A resolution, shows the occurrence of the cleavage reaction in the crystal, with the product peptides still held in the enzyme active site. The separation between the scissile carbon and nitrogen atoms is 2.67 A, which is shorter than a normal van der Waal separation, but it is much longer than a peptide bond length. The substrate is thus in a stage just past the G'Z intermediate described in Northrop's mechanism [Northrop DB (2001) Acc Chem Res 34:790-797]. Because the products are generated in situ, the structure, by extrapolation, can give insight into the mechanism of the cleavage reaction. Both oxygens of the generated carboxyl group form hydrogen bonds with atoms at the catalytic center: one to the OD2 atom of a catalytic aspartate and the other to the scissile nitrogen atom. The latter hydrogen bond may have mediated protonation of scissile nitrogen, triggering peptide bond cleavage. The inner oxygen atoms of the catalytic aspartates in the complex are 2.30 A apart, indicating a low-barrier hydrogen bond between them at this stage of the reaction, an observation not included in Northrop's proposal. This structure forms a template for designing mechanism-based inhibitors.

Bis(glycinium) oxalate: evidence of strong hydrogen bonding.

In the title 2:1 salt, 2C2H6NO2+.C2O4(2-), the glycine molecule is in the cationic form with a positively charged amino group and an uncharged carboxylic acid group. The doubly charged oxalate anion lies across a crystallographic inversion centre. One of the reasons why the 1:1 glycinium oxalate salt has a higher melting point than the title compound may be the difference in their hydrogen-bonding patterns. A database search for salts formed between amino acids or substituted amino acids and oxalic acid revealed that, in most of the structures, the conformation about the O=C-OH bond is synplanar. D-Tryptophan oxalate is the only example where the OH group of a semi-oxalate adopts an antiplanar conformation. The 2:1 stoichiometry seen in the present salt is observed only in the salts of DL-serine, DL-aspartic acid and betaine with oxalic acid.

1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin.

Under the selection pressure of drugs, mutations appear in HIV-1 protease even at the sites, which are conserved in the untreated individuals. Cysteine 95 is a highly conserved residue and is believed to be involved in regulation of HIV-1 protease. In some of the virus isolates from patients undergoing heavy treatment with anti-HIV protease drugs, C95F mutation has appeared. The present study reports 1.8A X-ray structure of C95M/C1095F double mutant of tethered HIV-1 protease dimer complexed with acetyl pepstatin. It is found that in this mutant, dimer interface has become more rigid and that the packing at the interface of terminal and core domains is altered. These alterations may be relevant to C95F mutation conferring drug resistance to HIV-1 protease.