Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity

Ketkar, Amit,Zafar, Maroof K.,Maddukuri, Leena,Yamanaka, Kinrin,Banerjee, Surajit,Egli, Martin,Choi, Jeong-Yun,Lloyd, R. Stephen,Eoff, Robert L. American Chemical Society 2013 Chemical research in toxicology Vol.26 No.2

<P>Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC<SUB>50</SUB> values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis–Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. <I>In silico</I> docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/crtoec/2013/crtoec.2013.26.issue-2/tx300392m/production/images/medium/tx-2012-00392m_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/tx300392m'>ACS Electronic Supporting Info</A></P>

Alleviating exercise-induced muscular stress using neat and processed bee pollen: oxidative markers, mitochondrial enzymes, and myostatin expression in rats

Sameer Ketkar,Atul Rathore,Amit Kandhare,Sathiyanarayanan Lohidasan,Subhash Bodhankar,Anant Paradkar,Kakasaheb Mahadik 한국한의학연구원 2015 Integrative Medicine Research Vol.4 No.3

Background: The current study was designed to investigate the influence of monofloral Indian mustard bee pollen (MIMBP) and processed monofloral Indian mustard bee pollen (PMIMBP) supplementation on chronic swimming exercise-induced oxidative stress implications in the gastrocnemius muscle of Wistar rats. Methods: MIMBP was processed with an edible lipid-surfactant mixture (Captex 355:Tween 80) to increase the extraction of polyphenols and flavonoid aglycones as analyzed by UV spectroscopy and high performance liquid chromatography-photo diode array. Wistar rats in different groups were fed with MIMBP or PMIMBP supplements at a dose of 100 mg/kg, 200 mg/kg and 300 mg/kg individually, while being subjected to chronic swimming exercise for 4 weeks (5 d/wk). Various biochemical [superoxide dismutase (SOD), glutathione (GSH), malonaldehyde (MDA), nitric oxide (NO), and total protein content], mitochondrial (Complex I, II, III, and IV enzyme activity), and molecular (myostatin mRNA expression) parameters were monitored in the gastrocnemius muscle of each group. Results: Administration of both MIMBP (300 mg/kg) and PMIMBP (100 mg/kg, 200 mg/kg, and 300 mg/kg) wielded an antioxidant effect by significantly improving SOD, GSH, MDA, NO, and total protein levels. Further MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) significantly improved impaired mitochondrial Complex I, II, III, and IV enzyme activity. Significant down-regulation of myostatin mRNA expression by MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) indicates a muscle protectant role in oxidative stress conditions. Conclusion: The study establishes the antioxidant, mitochondrial upregulatory, and myostatin inhibitory effects of both MIMBP and PMIMBP in exercise-induced oxidative stress conditions, suggesting their usefulness in effective management of exercise-induced muscular stress. Further, processing of MIMBP with an edible lipid-surfactant mixture was found to improve the therapeutic efficiency of pollen.

Human Rev1 polymerase disrupts G-quadruplex DNA

Eddy, Sarah,Ketkar, Amit,Zafar, Maroof K.,Maddukuri, Leena,Choi, Jeong-Yun,Eoff, Robert L. Oxford University Press 2014 Nucleic acids research Vol.42 No.5

<P>The Y-family DNA polymerase Rev1 is required for successful replication of G-quadruplex DNA (G4 DNA) in higher eukaryotes. Here we show that human Rev1 (hRev1) disrupts G4 DNA structures and prevents refolding <I>in vitro</I>. Nucleotidyl transfer by hRev1 is not necessary for mechanical unfolding to occur. hRev1 binds G4 DNA substrates with <I>K</I><SUB>d,DNA</SUB> values that are 4–15-fold lower than those of non-G4 DNA substrates. The pre-steady-state rate constant of deoxycytidine monophosphate (dCMP) insertion opposite the first tetrad-guanine by hRev1 is ∼56% as fast as that observed for non-G4 DNA substrates. Thus, hRev1 can promote fork progression by either dislodging tetrad guanines to unfold the G4 DNA, which could assist in extension by other DNA polymerases, or hRev1 can prevent refolding of G4 DNA structures. The hRev1 mechanism of action against G-quadruplexes helps explain why replication progress is impeded at G4 DNA sites in Rev1-deficient cells and illustrates another unique feature of this enzyme with important implications for genome maintenance.</P>

Potentiating antimicrobial efficacy of propolis through niosomal-based system for administration

Jay Patel,Sameer Ketkar,Sharvil Patil,James Fearnley,Kakasaheb Mahadik,Anant Paradkar 한국한의학연구원 2015 Integrative Medicine Research Vol.4 No.2

Background: Propolis is a multicomponent active, complex resinous substance collected by honeybees (Apis mellifera) from a variety of plant sources. This study was designed to improve the antimicrobial efficacy of propolis by engineering a niosomal-based system for topical application. Methods: Propolis was extracted in ethanol and screened for total polyphenol content. Propolis-loaded niosomes (PLNs) were prepared with varying concentrations of Span 60 and cholesterol. The PLNs were evaluated for physicochemical parameters, namely, vesicle size, entrapment efficiency, zeta potential, surface topography and shape, and stability, followed by screening for in vitro antimicrobial activity. The PLNs were formulated into propolis niosomal gel (PNG) using Carbopol P934 base and subjected to ex vivo skin deposition study. Results: The ethanolic extract of propolis had high polyphenolic content (270 ± 9.2 mg GAE/g). The prepared PLNs showed vesicle size between 294 nm and 427 nm, and the percent entrapment in the range of 50.62–71.29% with a significant enhancement in antimicrobial activity against Staphylococcus aureus and Candida albicans. Enhanced antimicrobial activity of PLNs was attributed to the ability of niosomes to directly interact with the bacterial cell envelop thereby facilitating the diffusion of propolis constituents across the cell wall. The formulated PNG exhibited a twofold better skin deposition due to improved retention of niosomes in the skin. Conclusion: The findings indicate that the engineering of a niosomal-based system for propolis enhanced its antimicrobial potential through topical application.