Biology subjects

Pillai, K.

Publications and source records attributed to Pillai, K..

4 recordsLinked to original sources

bioRxiv2025

Molecular Dynamics-Guided Design and Chemoproteomic Profiling of Covalent Kinase Activity Probes

Covalent small molecule activity probes can be powerful tools to interrogate protein function in native cellular environments. The design of family-wide activity probes requires an understanding of the molecular sources of general targeting potential and specificity to enable broad targeting of protein family members. Here, we developed and applied a multifaceted docking and molecular dynamics (MD) simulation pipeline to design and test cell-permeable covalent kinase activity probes from a set of hinge-binding pharmacophores. This computationally-guided approach yielded a new cell-active probe, K60P, which targets around 114 kinases across distinct kinase classes in live cells. Chemoproteomic profiling of this probe and a clinical candidate sharing the same indazole core, KW-2449, identified kinase and non-kinase target profiles that differ from recombinant protein assay profiles, underscoring the utility of native kinase profiling in situ. Biochemical studies with a model target kinase, ABL1, confirmed covalent labeling of the active site lysine across several kinase probes with distinct kinetics, as well as covalent labeling of key tyrosines in trans between ABL1 monomers. Finally, focused proteomics, kinetic modeling, and molecular dynamics simulations revealed that K60P, as well as the comparator probe XO44, preferentially engage with target kinases in their active, DFG-in conformations, which is driven by increasing population of reaction-ready small molecule conformation. These results together establish a computational and kinetic modeling framework for designing covalent activity probes and highlight the balance of target selectivity and kinetic efficiency as a key factor in determining their proteome-wide reactivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/683178v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ea3e5forg.highwire.dtl.DTLVardef@1a4e999org.highwire.dtl.DTLVardef@1e468c3org.highwire.dtl.DTLVardef@99e04e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Effect of nebulised BromAc(R) on rheology of artificial sputum: relevance to muco-obstructive respiratory diseases including COVID-19

Respiratory diseases such as cystic fibrosis, COPD, bronchiectasis asthma and COVID-19 are difficult to treat owing to viscous secretions in the airways that evade mucocilliary clearance. Since earlier studies have shown success with BromAc(R) as mucolytic agent for treating a rare disease known as pseudomyxoma peritonei (PMP), we tested the formulation on two gelatinous airway representative sputa models, in order to determine whether similar efficacy exist. The sputum (1.5 ml) lodged in an endotracheal tube was treated to aerosolised N-acetylcysteine, bromelain, or their combination (BromAc(R)) using a nebuliser with 6.0 ml of the agents in phosphate buffer saline, over 25 min. Controls received phosphate buffer saline. The dynamic viscosity was measured before and after treatment using a capillary tube method, whilst the sputum flow (ml/sec) was assessed using a 0.5 ml pipette. Finally, the sequestered agents (concentration) in the sputa after treatment were quantified using standard bromelain and N-acetylcysteine chromogenic assays. Results indicated that bromelain and N-acetylcysteine affected both the dynamic viscosities and pipette flow in the two sputa models, with changes in the former parameter having immense effect on the latter. BromAc(R) showed a greater rheological effect on both the sputa models compared to individual agents. Further, correlation was found between the rheological effects and the concentration of agents in the sputa. Hence, this study indicates that BromAc(R) may be used as a successful mucolytic for clearing airway congestion caused by thick mucinous immobile secretion, however further studies with patient sputum samples using aerosol BromAc(R) is warranted.

microbiology↗

bioRxiv2021

Bromelain and Acetylcysteine (BromAc) alone and in combination with Gemcitabine inhibits subcutaneous deposits of pancreatic cancer after intraperitoneal injection

ObjectiveGemcitabine (GEM) is commonly chosen for treating pancreatic cancer. However, its use is limited by toxicity. Earlier in vitro studies with GEM in combination with Bromelain (Brom) and Acetylcysteine (Ac) indicated a substantial reduction in IC50. Here, we investigated the efficacy and safety of Brom and Ac (BromAc) in the pancreatic cancer model in vivo. DesignBoth low dose and high dose studies for safety and efficacy of BromAc and GEM were conducted in nude mice. Body weight, wellbeing and tumor volume were monitored. At autopsy, tumor weight, tumor density, percentage of tumor necrosis, expression of Ki67 antigen, and immunohistological evaluation of vital organs were compared between the treatment groups. ResultsThe low and high doses of BromAc alone and with chemotherapy agents were safe. A very significant reduction in pancreatic tumor volume, weight, and ki67 were seen with BromAc therapy and was equal to treatment with GEM alone and better than treatment with 5-FU. In addition, tumor density was significantly reduced by BromAc. ConclusionThese encouraging results are the first in vivo evidence of the efficacy of BromAc in pancreatic cancer and provide some mechanistic leads.

cancer biology↗

bioRxiv2020

In vitro study of BromAc on SARS-CoV-2 spike and envelope proteins shows synergy and disintegration at modest concentrations

Background and objectivesSARS-CoV-2 infection is the cause of a worldwide pandemic, currently with limited therapeutic options. Whilst vaccines are at the forefront of the therapeutic initiative, drug repurposing remains a promising approach for SARS-CoV-2 treatment. BromAc (Bromelain & Acetylcysteine) has synergistic action against glycoproteins by the synchronous breakage of glycosidic linkages and disulfide bonds. The spike protein of SARS-CoV-2, formed of glycoprotein and disulfide bridges for stabilization, represents an attractive target as it is essential for binding to the ACE2 receptor in host cells present in nasal mucosa. We sought to determine the effect of BromAc on the Spike and Envelope proteins and its potential to reduce infectivity in host cells. DesignRecombinant Spike and Envelope proteins were treated by single agent and combination BromAc at 50 and 100 {micro}g/20mg/mL and analyzed by electrophoresis. Ultraviolet analysis of disulfide bond reduction was performed for both Spike and Envelope proteins after treatment with Acetylcysteine. In vitro whole virus culture inactivation of pre-treated wild type and an S1/S2 Spike mutant SARS-CoV-2 with BromAc from 25 to 250 {micro}g/20mg/mL was measured by cytopathic effect, cell lysis assay, and replication capacity by RT-PCR. ResultsRecombinant Spike and Envelope SARS-CoV-2 proteins were fragmented by BromAc at both 50 and 100 {micro}g/20mg/mL whilst single agents had minimal effect. Spike and Envelope protein disulfide bonds were reduced by Acetylcysteine. In vitro whole virus culture of both wild type and Spike mutant SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. ConclusionBromAc disintegrates SARS-CoV-2 Spike and Envelope proteins. In vitro tests on whole virus support this finding with inactivation of its replication capacity most strongly at 100 and 250 {micro}g/20mg/mL BromAc, even in Spike mutant virus. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent. Author SummaryThere is currently no suitable therapeutic treatment for early SARS-CoV-2 aimed to prevent disease progression. BromAc is under clinical development by the authors for mucinous cancers due to its ability to alter complex glycoproteins structure. The potential of BromAc on SARS-CoV-2 Spike and Envelope glycoproteins stabilized by disulfide bonds was examined and found to disintegrate recombinant Spike and Envelope proteins whilst reducing disulfide stabilizer bridges. BromAc also showed an inhibitory effect on wild-type and Spike mutant SARS-CoV-2 by inactivation of its replication capacity in vitro. Hence, BromAc may be an effective therapeutic agent for early SARS-CoV-2 infection, despite mutations, and even have potential as a prophylactic in people at high risk of infection.

microbiology↗