Byron J. Aguilar

Polyisoprenylation Potentiates the Inhibition of Polyisoprenylated Methylated Protein Methyl Esterase and the Cell Degenerative Effects of Sulfonyl Fluorides

The polyisoprenylation pathway incorporates a reversible step that metabolizes polyisoprenylated methylated proteins from the ester to the carboxylate form. Polyisoprenylated protein methyl transferase (PPMTase) catalyses the esterification whereas polyisoprenylated methylated protein methyl esterase (PMPMEase) hydrolyzes them. Significant changes in the balance between the two enzymes may alter polyisoprenylated protein function possibly resulting in disease. Previous studies show that PMPMEase is the serine hydrolase, Sus scrofa carboxylesterase. Its susceptibility to the nonspecific serine hydrolase inhibitor, phenylmethylsulfonyl fluoride (PMSF) paved the way for its use as a prototypical compound to design and synthesize a series of putative high affinity specific inhibitors of PMPMEase. Pseudo first-order kinetics revealed an over 680-fold increase in k(obs)/[I] values from PMSF (6 M(-1)-1s(-1)), S-phenyl (L-50, 180 M(-1)s(-1)), S-benzyl (L-51, 350 M(-1)s(-1)), S-trans, trans-farnesyl (L-28, 2000 M(-1)s(-1)), to S-trans-geranylated (L-23, 4100 M(-1)s(-1)) 2-thioethanesulfonyl fluorides. C10 S-alkyl substitution revealed a k(obs)/[I] value (1800 M(-1)s(-1)) that was 298 times greater than that for PMSF. The compounds induced the degeneration of human neuroblastoma SH-SY5Y cells with EC(50) values of 49, 130 and >1000 µM for L-28, L-23 and PMSF, respectively. The increased affinity with the polyisoprenyl derivatization is consistent with the observed substrate specificity and the reported hydrophobic nature of the active site. These results suggest that (1) PMPMEase is a key enzyme for polyisoprenylated protein metabolism, (2) regulation of its activity is essential for maintaining normal cell viability, (3) abnormal activities may be involved in degenerative diseases and cancers and (4) its specific inhibitors may be useful in combating cancers.

Polyisoprenylated Methylated Protein Methyl Esterase Is Both Sensitive to Curcumin and Overexpressed in Colorectal Cancer: Implications for Chemoprevention and Treatment

Inhibition of PMPMEase, a key enzyme in the polyisoprenylation pathway, induces cancer cell death. In this study, purified PMPMEase was inhibited by the chemopreventive agent, curcumin, with a K i of 0.3 μM (IC50 = 12.4 μM). Preincubation of PMPMEase with 1 mM curcumin followed by gel-filtration chromatography resulted in recovery of the enzyme activity, indicative of reversible inhibition. Kinetics analysis with N-para-nitrobenzoyl-S-trans,trans-farnesylcysteine methyl ester substrate yielded K M values of 23.6 ± 2.7 and 85.3 ± 15.3 μM in the absence or presence of 20 μM curcumin, respectively. Treatment of colorectal cancer (Caco2) cells with curcumin resulted in concentration-dependent cell death with an EC50 of 22.0 μg/mL. PMPMEase activity in the curcumin-treated cell lysate followed a similar concentration-dependent profile with IC50 of 22.6 μg/mL. In colorectal cancer tissue microarray studies, PMPMEase immunoreactivity was significantly higher in 88.6% of cases compared to normal colon tissues (P < 0.0001). The mean scores ± SEM were 91.7 ± 11.4 (normal), 75.0 ± 14.4 (normal adjacent), 294.8 ± 7.8 (adenocarcinoma), and 310.0 ± 22.6 (mucinous adenocarcinoma), respectively. PMPMEase overexpression in colorectal cancer and cancer cell death stemming from its inhibition is an indication of its possible role in cancer progression and a target for chemopreventive agents.

Inhibition of Polyisoprenylated Methylated Protein Methyl Esterase by Synthetic Musks Induces Cell Degeneration

Synthetic fragrances are persistent environmental pollutants that tend to bioaccumulate in animal tissues. They are widely used in personal care products and cleaning agents. Worldwide production of Galaxolide and Tonalide are in excess of 4500 tons annually. Because of their widespread production and use, they have been detected in surface waters and fish in the US and Europe. Consumption of contaminated water and fish from such sources leads to bioaccumulation and eventual toxicity. Since fragrances and flavors bear structural similarities to polyisoprenes, it was of interest to determine whether toxicity by Galaxolide and Tonalide may be linked with polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition. A concentration‐dependent study of PMPMEase inhibition by Galaxolide and Tonalide as well as their effects on the degeneration of cultured cells were conducted. Galaxolide and Tonalide inhibited purified porcine liver PMPMEase with Ki values of 11 and 14 μM, respectively. Galaxolide and Tonalide also induced human cancer cell degeneration with EC50 values of 26 and 98 μM (neuroblastoma SH‐SY5Y cells) and 58 and 14 μM (lung cancer A549 cells), respectively. The effects on cell viability correlate well with the inhibition of PMPMEase activity in the cultured cells. Molecular docking analysis revealed that the binding interactions are most likely between the fragrance molecules and hydrophobic amino acids in the active site of the enzyme. These results appear to suggest that the reported neurotoxicity of these compounds may be associated with their inhibition of PMPMEase. Exposure to fragrances may pose a significant risk to individuals predisposed to developing degenerative disorders.

Abstract B89: Polyisoprenylated cysteinyl amide inhibitors of PMPMEase inhibit pancreatic cancer cell viability and migration: Implications for pancreatic cancer therapy

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly neoplasms due to lack of effective targeted therapies. This vacuum stems from the fact that over 90% of pancreatic cancer cases harbor the K-Ras mutation which has been very challenging to drug. The numerous efforts to drug oncogenic Ras have involved inhibiting the post-translational modifications that are essential to its proliferative activity. These drug discovery efforts have largely ignored a critical step in the metabolism of Ras and related proteins involving polyisoprenylated methylated protein methyl esterase (PMPMEase). The objectives of these studies were to test the hypothesis that PMPMEase hyperactivity promotes pancreatic cancer progression. The potential for polyisoprenylated cysteinyl amide inhibitors (PCAIs) of PMPMEase as potential anticancer agents was evaluated against PDAC cell lines MIAPaCa-2 with K-Ras mutation and BxPC-3 with wild type K-Ras for their effects on cell viability, migration, and cytoskeletal organization. The PCAIs inhibited the viability of MIAPaCa-2 and BxPC-3 cells with 48 h EC50 values as low as 1.9 and 3.2 µM, respectively. Erlotinib and Salirasib (FTS) were ineffective at concentrations in excess of 20 µM. The PCAIs also inhibited MIAPaCa-2 cell migration by up to 50% at 0.5 µM and disrupted F-actin organization at 5 µM. Further investigations showed that at the EC50 concentrations the PCAIs caused the pancreatic cancer cells to die by apoptosis. The cell cycle analysis for MIAPaCa-2 cells showed that the PCAIs block the cells growth at G0/G1 phase. The results showed that the PCAIs are over 10-fold more effective than Salirasib and Erlotinib against PDAC cell viability. Furthermore, their effects against cell migration and actin filament organization suggest their potential for treating tumor growth and metastasis in not only PDAC but other cancers with oncogenic Ras. Citation Format: Augustine T. Nkembo, Byron J. aguilar, Randolph Duverna, Felix Amissah, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors of PMPMEase inhibit pancreatic cancer cell viability and migration: Implications for pancreatic cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B89.

Abstract 2035: Targeting the hyperactive polyisoprenylated monomeric G-proteins functions in pancreatic cancer

Pancreatic cancer (PC) incidence is a major healthcare problem as the five-year survival rate remains below 5%. Over 90% of PC cases harbor the hyperactive K-Ras mutant protein. Polyisoprenylation processes facilitate the proper localization and signaling of K-Ras whose gain-in-function mutations drive uncontrolled cell survival, differentiation and proliferation. Efforts to target and disrupt this pathway and signaling for cancer therapy have been unsuccessful. The purpose of this study was to determine the effects of polyisoprenylated cysteinyl amide inhibitors (PCAIs) as a potential novel class of small molecules for PC therapy. The effects of the PCAIs on cell viability, proliferation, survival, apoptosis, migration, colony formation and cytoskeletal F-actin organization in four human PC cell lines representing wild type K-Ras (BxPC-3), Mutant K-Ras (MIAPaCa-2 and Panc10.05) and mucins (HPAF-II) were examined. PCAIs induced apoptosis with EC50 values ranging from 2.4 to 5.1, 2.0 to 7.3, 1.8 to 3.3 and 7.0 to 33 μM in MIAPaCa-2, BxPC-3, Panc10.05 and HPAF-II cells, respectively. At 0.2 μM, PCAIs arrested MIAPaCa-2 cells at the G0/G1 phase and significantly inhibited cell migration in the wound-healing assay. MIAPaCa-2 cells transfected with a plasmid expressing a protein that fluoresces red when interacting with F-actin were used to determine the PCAIs effects on F-actin organization. Treatment with PCAIs disrupted the F-actin structures causing the cells to shrink and become rounded upon treatment with PCAIs concentrations as low as 0.2 μM. The ability of PCAIs to disrupt these biological phenomena in pancreatic cancer cells shows that PCAIs may halt progression in tumors with the hyperactive K-Ras mutations. Citation Format: Augustine T. Nkembo, Olufisayo Salako, Rosemary Poku, Tryphon Mazu, Byron Aguilar, Hernan Flores-Rozas, Nazarius S. Lamango. Targeting the hyperactive polyisoprenylated monomeric G-proteins functions in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2035. doi:10.1158/1538-7445.AM2015-2035

Polyisoprenylated methylated protein methyl esterase as a putative drug target for androgen-insensitive prostate cancer

Prostate cancer (CaP) is the most frequently diagnosed cancer in US men, with an estimated 236,590 new cases and 29,720 deaths in 2013. There exists the need to identify biomarkers/therapeutic targets for the early/companion diagnosis and development of novel therapies against the recalcitrant disease. Mutation and overexpression-induced abnormal activities of polyisoprenylated proteins have been implicated in CaP. Polyisoprenylated methylated protein methyl esterase (PMPMEase) catalyses the only reversible and terminal reaction of the polyisoprenylation pathway and may promote the effects of G proteins on cell viability. In this review, the potential role of PMPMEase to serve as a new drug target for androgen-insensitive CaP was determined. Specific PMPMEase activities were found to be 3.5- and 4.5-fold higher in androgen-sensitive 22Rv1 and androgen-dependent LNCaP and 1.5- and 9.8-fold higher in castration-resistant DU 145 and PC-3 CaP cells compared to normal WPE1-NA22 prostate cells. The PMPMEase inhibitor, L-28, induced apoptosis with EC50 values ranging from 1.8 to 4.6 μM. The PMPMEase activity in the cells following treatment with L-28 followed a similar profile, with IC50 ranging from 2.3 to 130 μM. L-28 disrupted F-actin filament organisation at 5 μM and inhibited cell migration 4-fold at 2 μM. Analysis of a CaP tissue microarray for PMPMEase expression revealed intermediate, strong, and very strong staining in 94.5% of the 92 adenocarcinoma cases compared to trace and weak staining in the normal and normal-adjacent tissue controls. The data are an indication that effective targeting of PMPMEase through the development of more potent agents may lead to the successful treatment of metastatic CaP.

Abstract LB-14: Polyisoprenylated cysteinyl amides as targeted small molecule anti-Ras therapies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Pancreatic cancer harbors the highest reported rates of K-Ras mutations, lacks effective therapies and is the most challenging to treat. It is thus the most deadly neoplasm with a 5-year survival rate of only 6%. We reveal here that polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in 93% of pancreatic duct adenocarcinoma. We further present novel compounds with the potential to serve as effective targeted therapies for pancreatic and other cancers with hyperactive growth signaling pathways mediated by Ras and related G-proteins. The design and synthesis of the polyisoprenylated cysteinyl amide inhibitors (PCAIs) of PMPMEase incorporate three key elements to obtain compounds to selectively disrupt polyisoprenylation-mediated protein-protein interactions; the farnesyl group for high affinity interactions, a substituted amide bioisostere of the scissile ester bond of the endogenous substrates and an ionizable appendage group designed to mitigate the excessive hydrophobicity of the farnesyl cysteinyl amide that constitutes the pharmacophore. The PCAIs inhibited PMPMEase with Ki values ranging from 3.7 to 20 μM. The 48 h EC50 values for pancreatic cancer Mia PaCa-2 and BxPC-3 cell lines were as low as 1.9 μM while Salirasib (farnesylthiosalicylic acid, FTS) and farnesylthiosalicylamide were not effective even at 20 μM. The PCAIs thus have the potential to serve as a novel class of targeted therapies for cancers with hyperactive G-proteins and the growth factor receptors whose signals they transmit. Citation Format: Nazarius S. Lamango, Byron J. Aguilar, Augustine T. Nkembo, Randolph Duverna, Felix Amissah, Rosemary A. Poku, Seth Y. Ablordeppey. Polyisoprenylated cysteinyl amides as targeted small molecule anti-Ras therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-14. doi:10.1158/1538-7445.AM2014-LB-14

Abstract 4357: Polyisoprenylated methylated protein methyl esterase as a potential therapeutic target and biomarker in lung cancer.

PURPOSE: Excessive epidermal growth factor receptor (EGFR) signaling and/or hyperactivities of polyisoprenylated monomeric G-proteins of the Ras superfamily are frequently associated with various human cancers including lung cancer. While mutations or overexpression of these proteins are responsible for their tumorigenic effects, secondary modifications of the monomeric G-proteins involving polyisoprenylation and eventual methylation are required for their normal as well as the pathological activities. Despite the use of anti-EGFR targeted therapies, prognosis is still poor for patients whose tumors harbor the defective receptor. The need to find novel biomarkers for early/companion diagnosis and targets for the development of novel therapies remains strong. Since polyisoprenylated methylated protein methyl esterase (PMPMEase) is a critical enzyme of the polyisoprenylation pathway whose inhibition causes cancer cell death, the objective was to determine whether it is overexpressed and/or hyperactive in lung cancers in which case, it might serve as a possible diagnostic biomarker or drug target. METHODS: Tissue microarrays (TMAs) consisting of 416 cores from 416 separate normal lung and lung cancer cases were analyzed for PMPMEase expression. Also, PMPMEase activities in fresh frozen lung cancer tissues were determined. PMPMEase expression and activities in normal lung fibroblasts (WI-38) as well as lung cancer A549 and H460 cells were also determined. The effect of specific PMPMEase inhibition with L-28 on cell viability was also tested. RESULTS: Significantly more intense PMPMEase immunoreactivity was observed in 88.3% of the 416 cases in the lung cancer compared to the normal controls (p CONCLUSION: These results implicate PMPMEase overexpression and hyperactivity in lung cancer progression, implying that PMPMEase could serve as a marker for diagnosis and target for lung cancer therapy. Citation Format: Felix Amissah, Randolph Duverna, Byron J. Aguilar, Rosemary A. Poku, Lamango S. Lamango. Polyisoprenylated methylated protein methyl esterase as a potential therapeutic target and biomarker in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4357. doi:10.1158/1538-7445.AM2013-4357

Abstract 418: Polyisoprenylated methylated protein methyl esterase overexpression in prostate cancer: implication in cancer metastasis.

Objectives: Prostate cancer is the most frequently diagnosed cancer among men in the U.S, with an estimated 241,740 new cases and 28,170 deaths for 2012. The need to identify biomarkers/therapeutic targets for the early/companion diagnosis and development of novel therapies against the recalcitrant form of prostate cancer is imperative. Mutation and overexpression-induced abnormal activities of polyisoprenylated proteins such as of the Ras and Rho families of monomeric G proteins have been implicated in prostate cancer. Also, signaling mediated by polyisoprenylated Rho family of GTPases through the actin cytoskeleton in migratory responses is necessary for cancer cell metastasis. Polyisoprenylated methylated protein methyl esterase (PMPMEase) catalyzes the only reversible and terminal reaction of the polyisoprenylation pathway. We hypothesized that PMPMEase activity may influence the effects of these G proteins on cell viability, metastasis and aggressiveness and thus may be of importance in the detection and treatment of prostate cancer. Method: Prostate cancer tissue microarray was probed for relative PMPMEase expression by immunohistochemistry. Specific activities of PMPMEase and the effect of its inhibition on viability of various prostate cancer cells were also studied. Effect of specific PMPMEase inhibitor L-28 on actin cytoskeleton organization was also determined by phalliodine staining of treated PC-3 cells. Results: PMPMEase specific activities were 4 and 6-fold higher in androgen-dependent 22Rv1 and LNCaP cells and 2- and 17-fold higher in androgen-independent PC-3 and DU 145 prostate cancer cells respectively compared to normal WPE1-NA22 prostate cells. Treatment of the cells for 72 h with L-28, a specific polyisoprenylated PMPMEase inhibitor induced apoptosis with EC50 values ranging from 1.8 to 4.6 μM. The PMPMEase activity of the different cell lines followed a similar profile with IC50 values ranging from 2.3 to 130 μM. L-28 also significantly disrupted the actin filament arrangement. Immunohistochemical analysis of PMPMEase expression revealed intermediate, strong and very strong staining in 94.5% of the 92 adenocarcinoma cases compared to only trace and weak staining in the normal and normal adjacent tissue controls. Conclusion: These results indicate that PMPMEase activity, immunoreactivity and inhibition could serve in the diagnosis and targeted therapeutic management of prostate cancer. Also inhibition of PMPMEase disturbs actin filament organization in cytoskeleton. Citation Format: Rosemary A. Poku, Felix Amissah, Randolph Duverna, Byron J. Aguilar, Nazarius S. Lamango. Polyisoprenylated methylated protein methyl esterase overexpression in prostate cancer: implication in cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 418. doi:10.1158/1538-7445.AM2013-418

Abstract 1843: Overexpression of polyisoprenylated methylated protein methyl esterase in triple negative breast cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Lack of targeted, more effective therapies for triple negative breast cancer (TNBC) has necessitated the continuous search for protein biomarkers and targets for novel therapeutic development. Polyisoprenylated monomeric G-proteins such as Ras and Rab are hyperactive in about 30% of cancers and have profound effects on cell growth. Inhibition of polyisoprenylated methylated protein methyl esterase (PMPMEase) causes the death of cancer cells thus suggesting that hyperactivity of PMPMEase may result in cancers. The current studies were to address the hypothesis that PMPMEase activity is elevated in breast cancer. PMPMEase activity was determined in breast tumors and normal adjacent tissues (NAT) in snap-frozen tissues from 10 breast cancer cases. Five- to over 20-fold increases in PMPMEase activity compared to the respective NATs were detected in 8 of the 10 cases. Specific activities ranging from 0.25 ± 0.02 to 1.58 ± 0.07 nmol/h/mg were detected in the tumors compared to 0.09 nmol/h/mg or less in the NATs. Breast cancer tissue microarrays (TMAs) consisting of 125 independent breast cancer cases and NATs were analyzed for PMPMEase expression by immunohistochemistry and scored for their immunoreactive staining intensities. Intermediate to very strong immunoreactivities were detected in the breast cancer TMA core slices compared to only trace staining in those of the NATs. The mean scores ± SEM ranged from 310 ± 21, 290 ± 7, 350 ± 15, 360 ± 18, 390 ± 14, 360 ± 29, 390 ± 13, 275 ± 10 to 65 ± 10 for TNBC, ER(+), ER(+)PR(+), ER(+)HER2(+), ER(+)PR(+)HER2(+), PR(+)HER2(+), HER2(+), PR(+) and NATs, respectively. These results show that PMPMEase overexpression in breast cancer may explain the increased enzymatic activity in the tumors. PMPMEase enzymatic activity and/or immunoreactivity may serve as diagnostic procedures for breast cancer. PMPMEase may also serve as valid target for anticancer drug development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1843. doi:1538-7445.AM2012-1843