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Papers by Sakshi Singh Tomar

Bioorganic & Medicinal Chemistry, 2015

The bat coronavirus HKU4 belongs to the same 2c lineage as that of the deadly Middle East Respira... more The bat coronavirus HKU4 belongs to the same 2c lineage as that of the deadly Middle East Respiratory Syndrome coronavirus (MERS-CoV) and shows high sequence similarity, therefore potentiating a threat to the human population through a zoonotic shift or 'spill over' event. To date, there are no effective vaccines or antiviral treatments available that are capable of limiting the pathogenesis of any human coronaviral infection. An attractive target for the development of anti-coronaviral therapeutics is the 3C-like protease (3CL pro), which is essential for the progression of the coronaviral life cycle. Herein, we report the screening results of a small, 230-member peptidomimetic library against HKU4-CoV 3CL pro and the identification of 43 peptidomimetic compounds showing good to excellent inhibitory potency of HKU4-CoV 3CL pro with IC 50 values ranging from low micromolar to sub-micromolar. We established structure-activity relationships (SARs) describing the important ligand-based features required for potent HKU4-CoV 3CL pro inhibition and identified a seemingly favored peptidic backbone for HKU4-CoV 3CL pro inhibition. To investigate this, a molecular sub-structural analysis of the most potent HKU4-CoV 3CL pro inhibitor was accomplished by the synthesis and testing of the lead peptidomimetic inhibitor's sub-structural components, confirming the activity of the favored backbone (22A) identified via SAR analysis. In order to elucidate the structural reasons for such potent HKU4-CoV 3CL pro inhibition by the peptidomimetics having the 22A backbone, we determined the X-ray structures of HKU4-CoV 3CL pro in complex with three peptidomimetic inhibitors. Sequence alignment of HKU4-CoV 3CL pro , and two other lineage C Betacoronaviruses 3CL pro 's, HKU5-CoV and MERS-CoV 3CL pro , show that the active site residues of HKU4-CoV 3CL pro that participate in inhibitor binding are conserved in HKU5-CoV and MERS-CoV 3CL pro. Furthermore, we assayed our most potent HKU4-CoV 3CL pro inhibitor for inhibition of HKU5-CoV 3CL pro and found it to have sub-micromolar inhibitory activity (IC 50 = 0.54 ± 0.03 lM). The X-ray structures and SAR analysis reveal critical insights into the structure and inhibition of HKU4-CoV 3CL pro , providing fundamental knowledge that may be exploited in the development of anti-coronaviral therapeutics for coronaviruses emerging from zoonotic reservoirs.

Journal of Biological Chemistry, 2015

Background: 3CL pro protease is required for coronaviral polyprotein processing and is only activ... more Background: 3CL pro protease is required for coronaviral polyprotein processing and is only active as a dimer. Results: MERS-CoV 3CL pro is a weakly associated dimer requiring ligand binding for dimer formation. Conclusion: Ligand-induced dimerization is a key mechanism for regulating the enzymatic activity of MERS-CoV 3CL pro during polyprotein processing. Significance: Activation via ligand-induced dimerization may add complexity for the development of MERS-CoV 3CL pro inhibitors as antivirals. All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the ␤-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL pro) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL pro from other ␤-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL pro is less efficient at processing a peptide substrate due to MERS-CoV 3CL pro being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL pro enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL pro is a weakly associated dimer (K d ϳ52 M) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL pro were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL pro undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å , reveal remarkable structural similarity of the dimer interface with 3CL pro from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL pro dimerization. Activation of MERS-CoV 3CL pro through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL pro inhibitors as antiviral agents. * This work was supported, in whole or in part, by National Institutes of Health Grants AI08508 (to A. D. M.) and AI026603 (to A. D. M. and M. R. D.). This work was also supported by the Walther Cancer Foundation (to A. D. M.). All authors reviewed the results and approved the final version of the manuscript. The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (codes 4RSP and 4YLU) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Donald School Journal of Ultrasound in Obstetrics & Gynecology, 2009

The central nervous system is probably the first organ system to be investigated in utero by diag... more The central nervous system is probably the first organ system to be investigated in utero by diagnostic ultrasound. Anencephaly was the first congenital anomaly to be recognized by this technique before viability. Since then, the investigation of the fetal neural axis has steadily remained a central issue of antenatal sonography. Such an interest is explained by a number of reasons. CNS anomalies are frequent and often have a severe prognosis. In many cases they have a genetic background: consequently a large number of couples are at risk and warrant antenatal diagnosis. Modern high resolution ultrasound equipment yields a unique potential in evaluating normal and abnormal anatomy of the fetal neural axis beginning at very early stages of development.

Ultrasound in Medicine & Biology, 2009

A high frequency test object was manufactured by moulding a series of wall-less anechoic pipe str... more A high frequency test object was manufactured by moulding a series of wall-less anechoic pipe structures in a block of agar based TMM with pipe diameters 0.045-1.5mm. Each probe was scanned over the surface of the test object. The vertical depth range over which each pipe could be visualised was determined, and plotted as a function of the reciprocal of pipe diameter. The Resolution Integral was calculated for each transducer by measuring the area under the curve obtained by scanning each pipe. Characteristic Resolution (Dr) and Depth of Field (Lr) were also determined from these measurements. Results: Pipe structures with diameters down to 45 micron were successfully manufactured within a novel test object and imaged using probes with frequencies from 12-60MHz. Resolution Integrals ranged from 22 (Visualsonics 704 probe 40MHz) to 75 (Philips L17-5 probe) with corresponding Lr/Dr of 4.1 mm/190m and 50mm/670 m respectively. Conclusions: The extended Edinburgh Pipe Phantom was able to successfully determine imaging characteristics of ultrasound probes up to 60MHz.

mBio, 2014

Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbre... more Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airwa...

Journal of Virology, 2013

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle Ea... more Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-H...

$Research paper thumbnail of Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin$

Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009

$Research paper thumbnail of Crystallization and preliminary X-ray diffraction studies of the lipopolysaccharide core biosynthetic enzyme ADP- L -glycero- D -mannoheptose 6-epimerase from Escherichia coli K-12$

Acta Crystallographica Section D Biological Crystallography, 1999