Corresponding author (NCNPR): Premalatha Balachandran, prembala@olemiss.eduhttps://egrove.olemiss.edu/pharm_annual_posters_2022/1002/thumbnail.jp

Avula, Bharathi

Balachandran, Premalatha

Bennett, Maria M.

Kumarihamy, Mallika

Muhammad, Ilias

Nanayakkara, N. P. Dhammika

Tripathi, Siddharth

Wang, Mei

Zhang, Jin

Zhao, Jianping

English

eGrove (Univ. of Mississippi)

University of Mississippi eGrove Annual Poster Session 2022 Annual Poster Session 10-11-2022 Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways Premalatha Balachandran University of Mississippi, prembala@olemiss.edu Mallika Kumarihamy University of Mississippi Siddharth Tripathi University of Mississippi Bharathi Avula University of Mississippi Jianping Zhao University of Mississippi See next page for additional authors Follow this and additional works at: https://egrove.olemiss.edu/pharm_annual_posters_2022 Recommended Citation Balachandran, Premalatha; Kumarihamy, Mallika; Tripathi, Siddharth; Avula, Bharathi; Zhao, Jianping; Wang, Mei; Bennett, Maria M.; Zhang, Jin; Nanayakkara, N. P. Dhammika; and Muhammad, Ilias, "Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways" (2022). Annual Poster Session 2022. 3. https://egrove.olemiss.edu/pharm_annual_posters_2022/3 This Book is brought to you for free and open access by the Annual Poster Session at eGrove. It has been accepted for inclusion in Annual Poster Session 2022 by an authorized administrator of eGrove. For more information, please contact egrove@olemiss.edu. Authors Premalatha Balachandran, Mallika Kumarihamy, Siddharth Tripathi, Bharathi Avula, Jianping Zhao, Mei Wang, Maria M. Bennett, Jin Zhang, N. P. Dhammika Nanayakkara, and Ilias Muhammad This book is available at eGrove: https://egrove.olemiss.edu/pharm_annual_posters_2022/3 Inhibitory activity of Machaeridiol-based Novel anti-MRSA and anti-VRE compounds and their profiling for Cancer related signaling pathways*Premalatha Balachandran1,  Mallika Kumarihamy1, Siddharth Tripathi1, Bharathi Avula1, Jianping Zhao1,  Mei Wang2, Maria M. Bennett1, Jin Zhang1, NP Dhammika Nanayakkara1, Ilias Muhammad11National Center for Natural Products Research,  Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, MS 38677, USA.3Natural Products Utilization Unit, Agricultural Research Service, U.S. Department of Agriculture, University, MS 38677, USAMETHODSAntimicrobial assay: Antimicrobial assays were carried out using a published method (10). All organisms were obtained from the American Type Culture Collection (Manassas, VA, USA). These included Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 90113, Aspergillus fumigatus ATCC 204305, methicillin-resistant Staphylococcus aureus ATCC 1708 (MRSA), Escherichia coli ATCC 2452, Pseudomonas aeruginosa ATCC BAA-2018, Klebsiella pneumonia ATCC 2146 and vancomycin resistant Enterococcus faecium (VRE) ATCC 700221. Briefly, the antimicrobial activity was determined through a high throughput screening assay performed in 384 well plate. Appropriate drug controls for bacteria and fungi were in-cluded in each assay. The concentration of compound/fraction responsible for 50% growth inhibition (IC50) was calculated using XLfit 4.2 software (IDBS, Alameda, CA) using fit model 201. Minimum inhibitory concentration (MIC) was defined as the lowest test concentration that affords no visual growth. Susceptibility testing was performed using a modified version of the CLSI (formerly NCCLS) method [4,5].Antimicrobial combination study by Checkerboard method: The combination study of the compounds was carried out in MRSA using a standard Checkerboard method by Norden et al. 1979 . Test samples were dissolved in 100% DMSO to the desired concentrations, and serially-diluted (1:2) with 20% DMSO/saline. For checkerboard, compound 5, 17 and machaeriol C (ie., previously isolated from Machaerium sp. (8, 9) were tested in the range of 2.5–0.039 µg/mL. Inocula was prepared in cation-adjusted Mueller- Hinton broth to afford 5 x 105 colony forming units per mL. Samples were transferred to 96 well assay plates (10µL) in a checkerboard layout followed by inocula (180µL). The MIC of each antimicrobial agent alone and in combination was determined after incubation at 35C for 24h. The fractional inhibitory concentration (FIC) was calculated by using the following formula: FIC = [A*]/[A] + [B*]/[B], where [A*] is MIC of compound A in the presence of compound B, [A] is MIC of compound A alone, [B*] is MIC of compound B in the presence of compound A, and [B] is MIC of compound B alone. FIC: 0.5 = synergistic; 0.51-1.0 = additive; 1.1-2.0 = indifferent; >2.0 = antagonistic [6,7,8].Transcriptional Reporter Assays: T98G glioblastoma multiforme cells from ATCC were plated in white opaque 384 well plates at a density of 4300 cells/well in 30 μL of growth medium (DMEM with 10% FBS and 1% Pen/Strep). Next day, the medium was aspirated and replaced with DMEM containing 10% FBS. The cells were transfected with respective plasmids using X-tremeGENE HP transfection reagent (Roche). Luciferase vectors used in this assay are summarized in (Table, Supporting Information). After 24 h of transfection, the test agents were added to the transfected cells, followed 30min later by an inducing agent (IL-6 for Stat 3, TGF-  for Smad, m-wnt3a for Wnt and PMA for AP-1, NFkB, E2F, Myc, ETS, Notch and Hedgehog). No inducer was added for FoXO, miR-21, Ras, AhR and pTK vector control. After 4 h or 6 h of induction, the cells were lysed by the addition of One-Glo luciferase assay system (Promega). The light output was detected in a Glomax Multi+ detection system with Instinct Software (Promega). This luciferase assay determines if the test agent was able to inhibit the activation of cancer-related signaling pathways. In the case of FoxO, mi-R21, Ras and AhR enhanced luciferase activity by the test agents was assessed [9,10].This work was supported by the USDA ARS-SCA # 58-6060-6-015, in part funding from the University of Mississippi Medical Center Cancer Institute to PB and overhead funds from NCNPR, UM.Results from antimicrobial assaysv Machaeridiols A-C were previously isolated from Macheriumpers.v Based on 5,6-seco- HHDBP machaeridiols A and C, new trans-benzopyan, styryl and resveratrol analogs 1-17 were synthesized (Fig. 1). v All these compounds were subjected to anti-microbial screens against a panel of microorganisms, including MRSA, vancomycin resistant Enterococci and Enterococcus faecium (VRE) and Cryptococcus neoformans to assess their IC50 values.v Compounds 4,6,9+10, 12 and 16 were found inactive (IC50 >20 µg/ml) (Table 1). v All compounds were inactive against vero cellsv Analogs from stemofuran A, pinosylvin and resveratrol legends showed strong to moderate IC50 values in the range of 0.91-8.04 and 1.17 -3.38 µg/ml against MRSA and VRErespectively.v Compounds 5,11,13,14 and 17 were selected for MIC determination based on their IC50 values of <3.0 µg/ml.v Among Stemofuran A (1-5 and 17), pinosylvin (6-14) and resveratrol (15 and 16) compounds 5,11,17 exhibited most potent activity against MRSA with IC50/MIC values of 1.18/2.50 µg/ml, 0.91/1.25 µg/ml and 0.95/1.25 µg/ml respectively.v These 3 analogs also found be effective against VRE with IC50/MIC of 2.27/2.50 µg/ml, 1.17/2.50 µg/ml, 2.28/2.50 µg/mlrespectively. v Among pinosylvin-geraniol analogs (12-14), compound 14 was found be active against MRSA and VRE with MIC of 5 µg/mlv The activities of 5,11 and 17 are similar to those of natural products machaeridiol C and A, but more potent than positive control meropenem.v In combination studies (checkboard assay), machaeriol C improved anti—MRSA activity of compound 17 as MIC decreased to 0.156 µg/ml. This combination also showed synergistic effect with a fractional inhibitory concentration value of (FIC) 0.5 µg/ml.v Compound 5 + machaeriol C displayed additive effect with a FIC value of 0.75 µg/ml. (Table 2)ABSTRACTThree unique 5,6-seco-hexahydrodibenzopyrans (seco-HHDBP) machaeridiols A–C, reported previously from Machaerium Pers., have displayed potent activities against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium and E. faecalis (VRE). In order to enrich pipeline of natural product- derived antimicrobial compounds, a series of novel machaeridiol based analogs (1-17) were prepared by coupling stemofuran, pinosylvin and resveratrol legends with monoterpene units R-(-)-α-phellandrene, (-)-p-mentha-2,8-diene-1-ol and geraniol, and their inhibitory activities profiled against MRSA ATCC 1708, VRE ATCC 700221, and cancer signalingpathways. Compounds 5 and 11 showed strong in vitro activities with MIC values of 2.5 and 1.25 μg/mL against MRSA, respectively, and 2.50 μg/mL against VRE, while geranyl analog 14 was found to be moderately active (MIC 5 μg /mL). Reduction of the double bonds of the monoterpene unit of compound 5 resulted in 17, which had the same antibacterial potency (MIC 1.25 and 2.50 μg/mL) as its parent, 5. Furthermore, a combination study between seco-HHDBP 17 and HHDBP machaeriol C displayed a synergistic effect with a fractional inhibitory concentrations (FIC) value of 0.5 against MRSA, showing a 4- fold decrease in MIC of both 17 and machaeriol C, while no such effect was observed between vancomycin and 17. Finally, compounds 1-17 were profiled against a panel of luciferase genes that assessed the activity of a complex cancer-related signaling pathways (i.e., transcription factors), using T98G glioblastoma multiforme cells. Among the compounds tested, the geranyl- substituted analog 14 exhibited strong inhibition against several signaling pathways, notably Smad, Myc, and Notch with IC50 values of 2.17, 1.86 and 2.15 μM, respectively. In contrast, the anti-MRSA compounds 5 and 17 were found to be inactive (IC50 >20 μM) across the panel of these cancer signaling pathways. MachaeridiolA and CDerivative Synthesis Analogs 1-17Anti-MRSA and Anti-CancerActivitiesPotential Lead Compounds (14 and 17)Fig. 2: Schematic representation of chemical reactions and in vitro analysisFig. 3: Schematic assay protocol of cancer-related signal transduction pathways using T98G glioblastoma multiforme cellsFig 1: Chemical structures of compounds isolated from Machaerium sp. and synthetic analogsCompoundsIC50 / MIC (μg/mL)*Methicillin-resistant           S. aureus (MRSA ATCC 1708)Vancomycin-resistant    E.  faecium (VRE ATCC 700221)Cryptococcus neoformans(ATCC 90113)1 3.24/ NT 2.49/ NT NA2 3.69/ NT 18.37/NT 16.74/ NT3 8.15/ NT  3.38/ NT  NA5 1.18/ 2.50** 2.27/2.50 2.15/ 2.507 8.04/NT   NA       NA8 16.51/NT      18.1/NT       NA11 0.95/ 1.25  2.28/ 2.50   0.95/ 2.5013 2.3/ 10.0   3.2/ 10.0    4.3/ NT14 3.0/ 5.0   2.3/ 5.0 1.5/ NT15 17.0/NT   17.8/NT     >20/NT17 0.91/ 1.25 1.17/ 2.50 1.92/ 5Machaeriol C# 0.69/1.25 1.16/2.5 NAMachaeridiol A# 1.03/2.5 0.72/2.5 15/NTMachaeridiol C# 0.38/1.25 0.72/1.25 25/NTMethicillin 2.2/50 >20/20 NTMeropenem 7.03/12.50 NA NAAmphotericin B NT NT 0.88/1.56CompoundMRSA (ATCC 1708)MIC (μg/mL)Concentration (μg/mL)Combination effectMIC (μg/mL)5 1.25 5+MC @ 0.625 MIC of 5 @ 0.312 ( 4X)Machaeriol C (MC) 1.25 MC+5 @ 0.625 MIC of MC @ 0.625 ( 2X) (FIC 0.75)17 0.625 17+MC @ 0.156 MIC of 17 @ 0.156 ( 4X)17+MC @ 0.312 MIC of 17 @ 0.312 ( 2X) Machaeriol C (MC) 1.25 MC+17 @ 0.156 MIC of MC @ 0.625 ( 2X) MC+17 @ 0.312 MIC of MC @ 0.312 ( 4X)  (FIC 0.50)  Vancomycin 1.00 - -- - 5 + Vancomycin          No effect            (FIC >1)- - 17 + Vancomycin         No effect            (FIC >1)Table 1: Antimicrobial activities of machaeridiol based synthetic analogs 1-3, 5,7-11,13-15 and 17Table 2: Combination study of compound 5 and 17 with machaeriol C by checkboard assay against MRSACompounds Stat3/IL-6Smad / TGF-betaAp-1/ PMANF-kB/ PMAE2F/PMAMyc/PMAEts/PMANotch/ PMA FoxOWnt/ m-wnt 3aHdghog/PMA miR-21 pTK Ras AhR7 8.99 10.5 - - <5 7.55 - 12.6 - 2.5 5.3 18.9 - - -8 4.9 3.5 10.25 7.66 4.5 5.7 7.3 <5 - 12.1 7.0 - - 7.0 ,2011 18.3 21 21 18.6 5.7 10.2 >20 10.59 - 15.9 13.79 - - 13.7813 6.55 9.95 10.89 16.9 4.9 4.8 11.44 <5 - 7.44 - 11.87 - 11.13 -14 4.33 2.17 4.45 4.8 4.43 1.86 4.7 2.12 13.7 9.11 8.35 - - 6.5 7.4Table 3: Activities of compounds 7,8,11,13 and 14 against cancer-related signaling pathways in T98G gliobastoma multiforme cellsResults from cancer signaling pathways assayØ Compounds 1-17 were screened against a panel of luciferase reporter genes that assesses the activity of a complex (cancer related signaling transduction pathways, using T98G glioblastoma multiforme cells. Ø Compounds 1-6, 9, 10, 12, 15-17 are considered inactive as their inhibition is less than 40% of the induction at the highest tested concentration of 20µM.Ø These compounds were excluded from further dose response studies and IC50 determination. (Results not shown)Ø Among the active compounds (7,8,11,13,14) shown in Table 3, the geraniol derivative of pinosylvin legend 14 exhibited strong specific activity against s Smad, Myc and Notch with IC50 values of 2.17, 1.86 and 2.12 µM respectively.Ø Compound 14 also showed between moderate inhibition in the range of 2.5 to 5 µM against Stat-3, AP-1, NF-kB, E2F, ETS pathways with mild inhibition against Foxo, Wnt, Hdghog, Ras and AhR pathways. Ø Surprisingly, the MRSA active stemofurans 5 and 17 and pinosylvin 11 analogs were found to be either inactive or weakly active against these signaling pathways. Cancer signaling assay details: Values are IC50 in µM that inhibited luciferase induction by 50%. The assay was run  This unique assay panel has a special feature of evaluating which pathways are sensitive (modulated) to a test agent. Each test agent was run at various concentrations and each concentration was run in duplicates. This multiplex assay offers many advantages for the simultaneous measurement and analysis of multiple pathways for any test agent, the intra-,inter-assay precision, accuracy and reproducibility are high for this method. The results are normalized to control vector which is pTK. The empty control vector allows us to see any non-specific cytotoxic effects of the test compounds on the cells. “-” means no activity detected.Antimicrobial assay details: Table 1:The primary (IC50) and secondary (MIC) assays were run separately and each executed in duplicates. NT-Not tested. NA-Not active. MRSA:Methicillin-resistant S.aureus, VRE: Vancomycin-resistant Enterococci. #Published values [1-3] for natural compounds are included for comparison with synthetic analogs. Table 2: 4x-4- fold reduction in MIC; 2x-2 -fold reduction in MIC. FIC: Fractional inhibitory concentrations.ConclusionsØ Several reports suggested a strong association of MRSA with a significant increase in cancer mortality. Ø Strong inhibition of compound 14 against several cancer signaling pathways, notably Smad, Myc and Notch (IC50<3µM) could possibly decrease the morbidity of cancer conditions (Fig 4).Ø The cross-talk between other pathways in response to compound 14 treatment (Stat 3, AP-1, NFkB, E2F and ETS with IC50 values in the range of 4-5µM reflect functional interactions and dependencies  (Fig 4).Ø These observations suggest that the compound 14 could be a potential lead molecule for targeted and combination therapies. Ø Thus, Compound 14 should be explored further for several cancers, more particularly in glioblastoma and in combination therapies against MRSA as well. References1. Muhammad, I et al., J. Nat. Prod. 2001, 64, 1322-1325.2. Muhammad, I et al., J. Nat. Prod. 2003, 66, 804-809.3. Muhammad, I et al., Molecules. 2020, 25, 6000.4. Gail, L. et al., Clinical and Laboratory Standards Institute.M24.A. 2003.5. Wikler MA. Clinical and Laboratory Standards Institute. M7 A7,2006.6. Norden, CW et al., J. Infect Dis. 1979. 140, 629–633.7. Hsieh, MH. et al., Diagn. Microbiol. Infect. Dis. 1993, 16, 343–349.8. Jacob, MR et al., J. Nat. Prod. 2003. 66, 1618–1622.9. Balachandran, P. et al., Molecules, 2021, 26, p.73510. Hamilton, KL. et al., J. Cancer Res. Clin. Oncol. 2021, 147, 445–457.Fig 4: Schematic representation of differential sensitivity of  compound 14 against signaling pathways*Molecules 2022, 27, 6604. https://doi.org/10.3390/molecules27196604.

Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways

https://egrove.olemiss.edu/cgi/viewcontent.cgi?article=1002&context=pharm_annual_posters_2022

Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways

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