A cell based assay using virus-like particles to screen AM type mimics for SARS-CoV-2 neutralisation.

A number of small molecule and protein therapeutic candidates have been developed in the last four years against SARS-CoV-2 spike. However, there are hardly a few molecules that have advanced through the subsequent discovery steps to eventually work as a therapeutic agent. This is majorly because of the hurdles in determining the affinity of potential therapeutics with live SARS-CoV-2 virus. Furthermore, affinity determined for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, at times, fails to mimic physiological conditions of the host-virus interaction. To bridge this gap between in vitro and in vivo methods of therapeutic agent screening, we report an improved screening protocol for therapeutic candidates using SARS-CoV-2 virus like particles (VLPs). To minimise the interference from the bulkier reporters like GPF in the affinity studies, a smaller hemagglutinin (HA) tag has been fused to one of the proteins of VLP. This HA tag serves as readout, when probed with fluorescent anti-HA antibodies. Outcome of this study sheds light on the lesser known virus neutralisation capabilities of AM type miniprotein mimics. Further, to assess the stability of SARS-CoV-2 spike - miniprotein complex, we have performed molecular dynamic simulations on the membrane embedded protein complex. Simulation results reveal extremely stable intermolecular interactions between RBD and one of the AM type miniproteins, AM1. Furthermore, we discovered a robust network of intramolecular interactions that help stabilise AM1. Findings from our in vitro and in silico experiments concurrently highlight advantages and capabilities of mimic based miniprotein therapeutics.

Design of human ACE2 mimic miniprotein binders that interact with RBD of SARS-CoV-2 variants of concerns.

The world of medicine demands from the research community solutions to the emerging problem of SARS-CoV-2 variants and other such potential global pandemics. With advantages of specificity over small molecule drugs and designability over antibodies, miniprotein therapeutics offers a unique solution to the threats of rapidly emerging SARS-CoV-2 variants. Unfortunately, most of the promising miniprotein binders are de novo designed and it is not viable to generate molecules for each new variant. Therefore in this study, we demonstrate a method for design of miniprotein mimics from the interaction interphase of human angiotensin converting enzyme 2 (ACE2). ACE2 is the natural interacting partner for the SARS-CoV-2 spike receptor binding domain (RBD) and acts as a recognition molecule for viral entry into the host cells. Starting with ACE2 N-terminal triple helix interaction interphase, we generated more than 70 miniprotein sequences. Employing Rosetta folding and docking scores we selected 10 promising miniprotein candidates amongst which 3 were found to be soluble in lab studies. Further, using molecular mechanics (MM) calculations on molecular dynamics (MD) trajectories we test interaction of miniproteins with RBD from various variants of concern (VOC). Presently, we report two key findings; miniproteins in this study are generated using less than 10 lab testing experiments, yet when tested through in-vitro experiments, they show submicro to nanomolar affinities towards SARS-CoV-2 RBD. Also in simulation studies, when compared with previously developed therapeutics, our miniproteins display remarkable ability to mimic ACE2 interphase; making them an ideal solution to the ever evolving problem of VOCs.Communicated by Ramaswamy H. Sarma.

Evolutionary conservation of protein dynamics: insights from all-atom molecular dynamics simulations of 'peptidase' domain of Spt16.

Protein function is encoded in its sequence, manifested in its three-dimensional structure, and facilitated by its dynamics. Studies have suggested that protein structures with higher sequence similarity could have more similar patterns of dynamics. However, such studies of protein dynamics within and across protein families typically rely on coarse-grained models, or approximate metrics like crystallographic B-factors. This study uses µs scale molecular dynamics (MD) simulations to explore the conservation of dynamics among homologs of ∼50 kDa N-terminal module of Spt16 (Spt16N). Spt16N from Saccharomyces cerevisiae (Sc-Spt16N) and three of its homologs with 30-40% sequence identities were available in the PDB. To make our data-set more comprehensive, the crystal structure of an additional homolog (62% sequence identity with Sc-Spt16N) was solved at 1.7 Å resolution. Cumulative MD simulations of 6 µs were carried out on these Spt16N structures and on two additional protein structures with varying degrees of similarity to it. The simulations revealed that correlation in patterns of backbone fluctuations vary linearly with sequence identity. This trend could not be inferred using crystallographic B-factors. Further, normal mode analysis suggested a similar pattern of inter-domain (inter-lobe) motions not only among Spt16N homologs, but also in the M24 peptidase structure. On the other hand, MD simulation results highlighted conserved motions that were found unique for Spt16N protein, this along with electrostatics trends shed light on functional aspects of Spt16N.Communicated by Ramaswamy H. Sarma.

Isolation and characterization of a recombinant class C acid phosphatase from Sphingobium sp. RSMS strain.

Tributyl phosphate (TBP) is extensively used in nuclear industry and is a major environmental pollutant. The mechanism for TBP degradation is not identified in any TBP-degrading bacteria. Here, we report identification of an acid phosphatase from Sphingobium sp. RSMS (Aps) that exhibits high specific activity towards monobutyl phosphate (MBP) and could be a terminal component of the TBP degradation process. A genomic DNA library of the bacteria was screened using a histochemical method which yielded 35 phosphatase clones. Among these, the clone that showed the highest MBP degradation was studied further. DNA sequence analysis showed that the genomic insert encodes a protein (Aps) which belongs to class C acid phosphatase. The recombinant Aps was found to be a dimer and hydrolysed MBP with a Kcat 68.1 ± 5.46 s- 1 and Km 2.5 mM ± 0.50. The protein was found to be nonspecific for phosphatase activity and hydrolyzed disparate organophosphates.

Autoproteolysis of Procerain and Procerain B mediated by structural changes.

Procerain (Pc) and Procerain B (PcB) are two latex proteases from Calotropis procera having potential applications in food and other industries. However, autolytic degradation of these proteases limits their potential use in industry. Nevertheless, basic mechanism underlying the autoproteolysis has not been detailed. In order to understand the same, we subjected the enzymes to various denaturing and activating conditions. The results showed that structural changes induced by different denaturing conditions trigger their autoproteolysis. We also observed differential response of Pc, PcB and other papain-like proteases towards autocatalysis in presence of reducing agent in-spite of sharing the same structural fold, including the number of disulfide bonds. The possible reason underlying this intriguing observation is also discussed. Further, present work establishes that structural changes in the proteases lead to autoproteolysis and the enzymes are stable unless they experience structural perturbation. These findings could thus be useful for their practical applications in industries.

Establishment and multiparametric-cytogenetic validation of 60Co-gamma-ray induced, phospho-gamma-H2AX calibration curve for rapid biodosimetry and triage management during radiological emergencies.

Exposure to ionizing radiation is unavoidable to our modern developing society as its applications are widespread and increasing with societal development. The exposures may be planned as in medical applications or may be unplanned as in occupational work and radiological emergencies. Dose quantification of planned and unplanned exposures is essential to make crucial decisions for management of such exposures. This study aims to establish ex-vivo dose-response curve for 60Co-gamma-ray induced gamma-H2AX-foci by immunofluorescence using microscopy and flowcytometry with human lymphocytes. This technique has the potential to serve as a rapid tool for dose estimation and triage application during small to large scale radiological emergencies and clinical exposures. Response curves were generated for the dose range 0-4 Gy (at 1, 2, 4, 8, 16, 24, 48, 72 and 96 h of incubation after irradiation) with microscopy and 0-8 Gy (at 2, 4, 8, 16 and 24 h of incubation after irradiation) with flow cytometry. These curves can be applied for dose reconstruction when post exposure sampling is delayed up to 96 h. In order to evaluate Minimum Detection Limit (MDL) of the assay, variation of background frequency of gamma-H2AX-foci was measured in 12 volunteers. To understand the application window of the assay, gamma-H2AX foci decay kinetics has been studied up to 96 h with microscopy and response curves were generated from 1 to 96 hours post exposure. Gamma-H2AX fluorescence intensity decay kinetics was also studied up to 96 h with flow cytometry and response curves were generated from 2 to 24 hours post irradiation. Established curves were validated with dose blinded samples and also compared with standard cytogenetic assays. An inter-comparison of dose estimates was made among gamma-H2AX assay, dicentric aberrations and reciprocal translocations for application window in various dose ranges and time of blood collection after exposures.

Machine learning classifiers aid virtual screening for efficient design of mini-protein therapeutics.

De novo design of mini-proteins (4-12 kDa) has recently been shown to produce new candidates for protein therapeutics. They are temperature stable molecules that bind to the drug target with high affinity for inhibiting its interactions. The development of mini-protein binders requires laboratory screening of tens of thousands of molecules for effective target binding. In this study we trained machine learning classifiers which can distinguish, with 90% accuracy and 80% precision, mini-protein binders from non-binding molecules designed for a particular target; this significantly reduces the number of mini protein candidates for experimental screening. Further, on the basis of our results we propose a multi-stage protocol where a small dataset (few hundred experimentally verified target-specific mini-proteins) can be used to train classifiers for improving the efficiency of mini-protein design for any specific target.

Two-domain aminopeptidase of M1 family: Structural features for substrate binding and gating in absence of C-terminal domain.

Zinc metallopeptidases of the M1 family (M1 peptidases) with unique metal binding motif HEXXH(X)18E regulate many important biological processes such as tumor growth, angiogenesis, hormone regulation, and immune cell development. Typically, these enzymes exist in three-domain [N-terminal domain (N-domain), catalytic domain, and C-terminal domain (C-domain)] or four-domain (N-domain, catalytic domain, middle domain, and C-domain) format in which N-domain and catalytic domain are more conserved. The C-domain plays important roles in substrate binding and gating. In this study we report the first structure of a two-domain (N-domain and catalytic domain) M1 peptidase at 2.05 Šresolution. Despite the lack of C-domain, the enzyme is active and prefers peptide substrates with large hydrophobic N-terminal residues. Its substrate-bound structure was determined at 1.9 Šresolution. Structural analyses supported by site directed mutagenesis and molecular dynamics simulations reveal structural features that could compensate for the lack of C-domain. A unique loop insertion (loop A) in the N-domain has important roles in gating and desolvation of active site. Three Arg residues of the catalytic domain are involved in substrate-binding roles typically played by positively charged residues of C-domain in other M1 peptidases. Further, its unique exopeptidase sequence motif, LALET, creates a more hydrophobic environment at the S1 subsite (which binds N-terminal residue of the substrate in aminopeptidases) than the more common GXMEN motif in the family. This leads to high affinity for large hydrophobic residues in the S1 subsite, which contributes towards efficient substrate binding in absence of C-domain.

Carboxypeptidase in prolyl oligopeptidase family: Unique enzyme activation and substrate-screening mechanisms.

Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. Proper annotation and knowledge of substrate specificity mechanisms in this family are highly valuable. Although endopeptidase, dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-Å resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. We observed that this residue is poised to bind substrate in the active structure and disrupts the catalytic triad in the inactive structure. The assembly of the active site is accompanied by the ordering of gating loops, which reduces the effective size of the oligomeric pore. This prevents the entry of larger peptides and constitutes a novel mechanism for substrate screening. Furthermore, we observed structural adaptations that enable its carboxypeptidase activity, with a unique loop and two arginine residues in the active site cavity orienting the peptide substrate for catalysis. Using these structural features, we identified homologs of this enzyme in the POP family and confirmed the presence of carboxypeptidase activity in one of them. In conclusion, we have identified a new type within POP enzymes that exhibits not only unique activity but also a novel substrate-screening mechanism.

Structure of Asp-bound peptidase E from Salmonella enterica: Active site at dimer interface illuminates Asp recognition.

Peptidase-E, a nonclassical serine peptidase, is specific for dipeptides with an N-terminal aspartate. This stringent substrate specificity remains largely unexplained. We report an aspartate-bound structure of peptidase-E at 1.83 Å resolution. In contrast to previous reports, the enzyme forms a dimer, and the active site is located at the dimer interface, well shielded from the solvent. Our findings further suggest that the stringent aspartate specificity of the enzyme is due to electrostatics and molecular complementarity in the active site. The new structural information presented herein may provide insights into the role of functionally important residues in peptidase-E.

Assessment of diabetic neuropathy with emission tomography and magnetic resonance spectroscopy.

Diabetic neuropathies (DNs) are nerve-damaging disorders associated with diabetes. They are commonly attributed to peripheral nerves and primarily affect the limbs of the patient. They cause altered sensitivity to external stimuli along with loss in balance and reflexes of the affected patient. DNs are associated with a variety of clinical manifestations including autonomic failure and are caused by poor management of blood sugar levels. Imaging modalities provide vital information about early physiological changes in DNs. This review summarizes contributions by various teams of scientists in developing imaging methods to assess physiological changes in DNs and ongoing clinical trials where imaging modalities are applied to evaluate therapeutic intervention in DNs. Development of PET, single photon emission computed tomography, and magnetic resonance spectroscopy methods over the past 20 years are reviewed in the diagnostic assessment of DNs. Abnormal radiotracer pharmacokinetics and neurometabolite spectra in affected organs confirm physiological abnormalities in DN. With the use of the Siemens Biograph mMR and GE Signa - 60 cm (PET/MRI scanner), simultaneous acquisition of physiological and anatomical information could enhance understanding of DNs and accelerate drug development.

Appraisal of Bleb Using Trio of Intraocular Pressure, Morphology on Slit Lamp, and Gonioscopy.

PURPOSE: The aim of this study was to assess bleb function using Wuerzburg bleb classification score (WBCS) for bleb morphology on slit lamp, intraocular pressure (IOP), and gonioscopy. METHODS: A total of randomly selected 30 eyes posttrabeculectomy were assessed for bleb function with the trio of bleb morphology, IOP, and gonioscopy. Bleb was assessed using the WBCS of 0-12 on slit lamp, IOP was assessed using applanation tonometry, and visualization of inner ostium and iridectomy were assessed using gonioscopy. Postoperative patients of less than six weeks were excluded from the study. RESULTS: The correlation between WBCS and the duration of trabeculectomy was found to be highly significant (P value = 0.029). The correlation of IOP with WBCS was also found to be strongly positive (P = 0.000). IOP was found to be highly associated with peripheral iridectomy (P = 0.000), internal window (P = 0.001), and bleb characteristics.

Is dexmedetomidine better than propofol and fentanyl combination in minor day care procedures? A prospective randomised double-blind study.

BACKGROUND AND AIMS: The growing popularity and trend of day care (ambulatory) anaesthesia has led to the development of newer and efficient drug regimen. We decided to evaluate the efficacy of two drug regimens namely dexmedetomidine and propofol with midazolam and fentanyl for moderate sedation characteristics in minor surgical procedures in terms of analgesia, intra-operative sedation, haemodynamic stability and side effects related. METHODS: Totally, 60 adult American Society of Anaesthesiologists class I-II patients posted for day care surgeries of duration <45 min divided into two groups; Group D, where dexmedetomidine loading dose at 1 µg/kg was administered over 10 min followed by maintenance infusion initiated at 0.6 µg/kg/h and titrated to achieve desired clinical effect with dose ranging from 0.2 to 0.7 µg/kg, Group P, where midazolam at 0.02 mg/kg and fentanyl at 2 µg/kg IV boluses were given followed by propofol infusion. Statistical analysis was done using student t-test, analysis of variance and Chi-square analysis. P < 0.05 was considered to be significant. RESULTS: Degree of sedation (Observer's Assessment of Activity and Sedation Scale ≤3) was comparable in both groups (P > 0.05). Rescue analgesia with fentanyl was needed in 30% patients of Group D compared to 17.63% patients of Group P (P < 0.05). The level of arousal was faster and better in Group D at 5 min after the procedure (P < 0.05). Haemodynamics were stable in Group D as with Group P patients (P < 0.005). Dry mouth reported by 16.67% patients. CONCLUSION: Dexmedetomidine can be a useful adjuvant rather than the sole sedative-analgesic agent during minor surgeries and be a valuable alternative to propofol in terms of moderate sedation, haemodynamic stability with minimal transient side effects.

A double-blind study on analgesic effects of fentanyl combined with bupivacaine for extradural labor analgesia.

BACKGROUND: The intermittent technique of labor extradural analgesia has been showing promising results over other techniques. This study was done to assess and compare the efficacy of two different doses of fentanyl mixed with low doses of bupivacaine in intermittent labor extradural analgesia. MATERIALS AND METHODS: 90 ASA grade I-II parturients in active labor with a cervical dilatation of 3-7 cm were randomly allocated to three different groups: Group A: 10 ml bupivacaine 0.125% + fentanyl 10 µg (1 µg/ml)Group B: 10 ml bupivacaine 0.125% + fentanyl 20 µg (2 µg/ml)Group C: 10 ml bupivacaine 0.125% (the control group) All patients were preloaded with 10-15 ml/kg Lactated Ringer's solution. Labor analgesia was maintained by intermittent boluses of the drug combination. RESULTS: The mean time of the onset of analgesia was significantly lower (P<0.05) and the duration of analgesia was significantly higher (P<0.01) in Group B when compared with Groups A and C (P<0.001). Patient satisfaction was considerably better in Group B (P<0.01). However, in both groups, the progression of labor was found to be slightly more prolonged than Group C. The level of the sensory and motor block was comparable in both the groups and was at the T8-T10 level; it was comparable and the level of motor blockade (Bromage score = 0, 1) in each group was also not significant (P>0.05). CONCLUSION: The addition of fentanyl (2 µg/ml) to bupivacaine 0.125% decreases the time of the onset of analgesia and increases the duration of analgesia and level of maternal satisfaction during labor as compared to fentanyl (1 µg/ml).