Activation of MAP Kinase Pathway by Polyisoprenylated Cysteinyl Amide Inhibitors Causes Apoptosis and Disrupts Breast Cancer Cell Invasion.

Prognoses for TNBC remain poor due to its aggressive nature and the lack of therapies that target its "drivers". RASA1, a RAS-GAP or GTPase-activating protein whose activity inhibits RAS signaling, is downregulated in up to 77% of TNBC cases. As such, RAS proteins become hyperactive and similar in effect to mutant hyperactive RAS proteins with impaired GTPase activities. PCAIs are a novel class of agents designed to target and disrupt the activities of KRAS and other G-proteins that are hyperactive in various cancers. This study shows the anticancer mechanisms of the PCAIs in two breast cancer cell lines, MDA-MB-468 and MDA-MB-231. PCAIs (NSL-YHJ-2-27) treatment increased BRAF phosphorylation, whereas CRAF phosphorylation significantly decreased in both cell lines. Moreover, the PCAIs also stimulated the phosphorylation of MEK, ERK, and p90RSK by 116, 340, and 240% in MDA-MB-468 cells, respectively. However, in MDA-MB-231 cells, a significant increase of 105% was observed only in p90RSK phosphorylation. Opposing effects were observed for AKT phosphorylation, whereby an increase was detected in MDA-MB-468 cells and a decrease in MDA-MB-231 cells. The PCAIs also induced apoptosis, as observed in the increased pro-apoptotic protein BAK1, by 51%, after treatment. The proportion of live cells in PCAIs-treated spheroids decreased by 42 and 34% in MDA-MB-468 and MDA-MB-231 cells, respectively, which further explains the PCAIs-induced apoptosis. The movement of the cells through the Matrigel was also inhibited by 74% after PCAIs exposure, which could have been due to the depleted levels of F-actin and vinculin punctate, resulting in the shrinkage of the cells by 76%, thereby impeding cell movement. These results show promise for PCAIs as potential therapies for TNBC as they significantly inhibit the hallmark processes and pathways that promote cell proliferation, migration, and invasion, which result in poor prognoses for breast cancer patients.

Lack of racial and ethnic diversity in lung cancer cell lines contributes to lung cancer health disparities.

Lung cancer is the leading cause of cancer death in the United States and worldwide, and a major source of cancer health disparities. Lung cancer cell lines provide key in vitro models for molecular studies of lung cancer development and progression, and for pre-clinical drug testing. To ensure health equity, it is imperative that cell lines representing different lung cancer histological types, carrying different cancer driver genes, and representing different genders, races, and ethnicities should be available. This is particularly relevant for cell lines from Black men, who experience the highest lung cancer mortality in the United States. Here, we undertook a review of the available lung cancer cell lines and their racial and ethnic origin. We noted a marked imbalance in the availability of cell lines from different races and ethnicities. Cell lines from Black patients were strongly underrepresented, and we identified no cell lines from Hispanic/Latin(x) (H/L), American Indian/American Native (AI/AN), or Native Hawaiian or other Pacific Islander (NHOPI) patients. The majority of cell lines were derived from White and Asian patients. Also missing are cell lines representing the cells-of-origin of the major lung cancer histological types, which can be used to model lung cancer development and to study the effects of environmental exposures on lung tissues. To our knowledge, the few available immortalized alveolar epithelial cell lines are all derived from White subjects, and the race and ethnicity of a handful of cell lines derived from bronchial epithelial cells are unknown. The lack of an appropriately diverse collection of lung cancer cell lines and lung cancer cell-of-origin lines severely limits racially and ethnically inclusive lung cancer research. It impedes the ability to develop inclusive models, screen comprehensively for effective compounds, pre-clinically test new drugs, and optimize precision medicine. It thereby hinders the development of therapies that can increase the survival of minority and underserved patients. The noted lack of cell lines from underrepresented groups should constitute a call to action to establish additional cell lines and ensure adequate representation of all population groups in this critical pre-clinical research resource.

Florida-California Cancer Research, Education and Engagement (CaRE2) Health Equity Center: Structure, Innovations, and Initial Outcomes.

INTRODUCTION: The Florida-California Cancer Research, Education, and Engagement (CaRE2) Health Equity Center is a triad partnership committed to increasing institutional capacity for cancer disparity research, the diversity of the cancer workforce, and community empowerment. This article provides an overview of the structure, process innovations, and initial outcomes from the first 4 years of the CaRE2 triad partnership. METHODS: CaRE2 serves diverse populations in Florida and California using a "molecule to the community and back" model. We prioritize research on the complex intersection of biological, environmental, and social determinants health, working together with scientific and health disparities communities, sharing expertise across institutions, bidirectional training, and community outreach. Partnership progress and outcomes were assessed using mixed methods and four Program Steering Committee meetings. RESULTS: Research capacity was increased through development of a Living Repository of 81 cancer model systems from minority patients for novel cancer drug development. CaRE2 funded 15 scientific projects resulting in 38 publications. Workforce diversity entailed supporting 94 cancer trainees (92 URM) and 34 ESIs (32 URM) who coauthored 313 CaRE2-related publications and received 48 grants. Community empowerment was promoted via outreaching to more than 3000 individuals, training 145 community cancer advocates (including 28 Community Scientist Advocates), and publishing 10 community reports. CaRE2 members and trainees together have published 639 articles, received 61 grants, and 57 awards. CONCLUSION: The CaRE2 partnership has achieved its initial aims. Infrastructure for translational cancer research was expanded at one partner institution, and cancer disparities research was expanded at the two cancer centers.

Polyisoprenylated cysteinyl amide inhibitors deplete singly polyisoprenylated monomeric G-proteins in lung and breast cancer cell lines.

Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are agents that mimic the essential posttranslational modifications of G-proteins. It is hypothesized that PCAIs work as anticancer agents by disrupting polyisoprenylation-dependent functional interactions of the G-Proteins. This study tested this hypothesis by determining the effect of the PCAIs on the levels of RAS and related monomeric G-proteins. Following 48 h exposure, we found significant decreases in the levels of KRAS, RHOA, RAC1, and CDC42 ranging within 20-66% after NSL-YHJ-2-27 (5 µM) treatment in all four cell lines tested, A549, NCI-H1299, MDA-MB-231, and MDA-MB-468. However, no significant difference was observed on the G-protein, RAB5A. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, after PCAIs treatment, migration, and invasion of A549 cells were inhibited by 72 and 70%, respectively while the levels of vinculin and fascin dropped by 33 and 43%, respectively. These findings implicate the potential role of PCAIs as anticancer agents through their direct interaction with monomeric G-proteins.

An Evaluation of the Anticancer Properties of SYA014, a Homopiperazine-Oxime Analog of Haloperidol in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a type of breast cancer associated with early metastasis, poor prognosis, high relapse rates, and mortality. Previously, we demonstrated that SYA013, a selective σ2RL, could inhibit cell proliferation, suppress migration, reduce invasion, and induce mitochondria-mediated apoptosis in MDA-MB-231 cell lines, although we were unable to demonstrate the direct involvement of sigma receptors. This study aimed to determine the anticancer properties and mechanisms of action of SYA014, [4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one oxime], an oxime analogue of SYA013, the contribution of its sigma-2 receptor (σ2R) binding, and its possible synergistic use with cisplatin to improve anticancer properties in two TNBC cell lines, MDA-MB-231 (Caucasian) and MDA-MB-468 (Black). In the present investigation, we have shown that SYA014 displays anticancer properties against cell proliferation, survival, metastasis and apoptosis in the two TNBC cell lines. Furthermore, a mechanistic investigation was conducted to identify the apoptotic pathway by which SYA014 induces cell death in MDA-MB-231 cells. Since SYA014 has a higher binding affinity for σ2R compared to σ1R, we tested the role of σ2R on the antiproliferative property of SYA014 with a σ2R blockade. We also attempted to evaluate the combination effect of SYA014 with cisplatin in TNBC cells.

Synthetic Optimization and MAPK Pathway Activation Anticancer Mechanism of Polyisoprenylated Cysteinyl Amide Inhibitors.

Abnormalities of the MAPK pathway play vital roles in cancer initiation and progression. RAS GTPases that are key upstream mediators of the pathway are mutated in 30% of human cancers. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed as potential targeted therapies against the RAS-driven cancers. The current study reports on the optimization of the PCAIs and the determination of their mechanisms of action in KRAS-mutant cancer cells. They display ClogP values ranging from 3.01 to 6.35, suppressing the viabilities of KRAS-mutant MDA-MB-231, A549, MIA PaCa-2, and NCI-H1299 cells in 2D and 3D cultures with EC50 values of 2.2 to 6.8, 2.2 to 7.6, 2.3 to 6.5 and 5.0 to 14 µM, respectively. When A549 cells were treated with the PCAIs, NSL-YHJ-2-27, for 48 h, no significant difference was observed in the levels of total or phosphorylated B- and C-Raf proteins. However, at 5 µM, it stimulated the phosphorylation of MEK1/2, ERK1/2, and p90RSK by 84%, 59%, and 160%, respectively, relative to controls. A non-farnesylated analog, NSL-YHJ-2-62, did not elicit similar effects. These data reveal that effects on the RAS-MAPK signaling axis most likely contribute to the anticancer effects of the PCAIs, possibly through the proapoptotic isoforms of p90RSK. The PCAIs may thus have the potential to serve the unmet therapeutic needs of patients with aberrant hyperactive G-protein signaling.

Polyisoprenylated Cysteinyl Amide Inhibitors: A Novel Approach to Controlling Cancers with Hyperactive Growth Signaling.

Aberrant activation of monomeric G-protein signaling pathways drives some of the most aggressive cancers. Suppressing these hyperactivities has been the focus of efforts to obtain targeted therapies. Polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in various cancers. Its inhibition induces the death of cancer cells that harbor the constitutively active K-Ras proteins. Furthermore, the viability of cancer cells driven by factors upstream of K-Ras, such as overexpressed growth factors and their receptors or the mutationally-activated receptors, is also susceptible to PMPMEase inhibition. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) were thus designed to target cancers with hyperactive signaling pathways involving the G-proteins. The PCAIs were, however, poor inhibitors of PMPMEase, with Ki values ranging from 3.7 to 20 µM. On the other hand, they inhibited cell viability, proliferation, colony formation, induced apoptosis in cells with mutant K-Ras and inhibited cell migration and invasion with EC50 values of 1 to 3 µM. HUVEC tube formation was inhibited at submicromolar concentrations through their disruption of actin filament organization. At the molecular level, the PCAIs at 2 to 5 µM depleted monomeric G-proteins such as K-Ras, RhoA, Cdc42 and Rac1. The PCAIs also deplete vinculin and fascin that are involved in actin organization and function while disrupting vinculin punctates in the process. These demonstrate a polyisoprenylation-dependent mechanism that explains the observed PCAIs' inhibition of the proliferative, invasive and angiogenic processes that promote both tumor growth and metastasis.

A Mechanistic Investigation on the Anticancer Properties of SYA013, a Homopiperazine Analogue of Haloperidol with Activity against Triple Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is one of the most malignant cancers associated with early metastasis, poor clinical prognosis, and high recurrence rate. TNBC is a distinct subtype of breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptors (HER2). Development of effective TNBC therapies has been limited partially due to the lack of specific molecular targets and chemotherapy involving different cytotoxic drugs suffers from significant side effects and drug-resistance development. Therefore, there is an unmet need for the development of novel and efficient therapeutic drugs with reduced side effects to treat TNBC. We have previously reported that certain analogues of haloperidol (a typical antipsychotic drug used for treating mental/mood disorders such as schizophrenia and bipolar disorder) suppress the viability of a variety of solid tumor cell lines, and we have identified 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluoro-phenyl)butan-1-one (SYA013) with such antiproliferative properties. Interestingly, unlike haloperidol, SYA013 shows moderate selectivity toward σ2 receptors. In this study, we explored the potential of SYA013 in modulating the important biological events associated with cell survival and progression as well as the mechanistic aspects of apoptosis in a representative TNBC cell line (MDA-MB-231). Our results indicate that SYA013 inhibits the proliferation of MDA-MB-231 cells in a concentration-dependent manner and suppresses cell migration and invasion. Apoptotic studies were also conducted in MDA-MB-468 cells (cells derived from a 51-year old Black female with metastatic adenocarcinoma of the breast.). In addition, we have demonstrated that SYA013 induces MDA-MB-231 cell death through the intrinsic apoptotic pathway and may suppress tumor progression and metastasis. Taken together, our study presents a mechanistic pathway of the anticancer properties of SYA013 against TNBC cell lines and suggests a potential for exploring SYA013 as a lead agent for development against TNBC.

Polyisoprenylated Cysteinyl Amide Inhibitors Deplete K-Ras and Induce Caspase-dependent Apoptosis in Lung Cancer Cells.

BACKGROUND: Non-small cell lung cancers (NSCLC) harboring mutation-induced dysregulation of Ras signaling present some of the most difficult-to-manage cases, since directly targeting the constitutively active mutant Ras proteins has not resulted in clinically useful drugs. Therefore, modulating Ras activity for targeted treatment of cancer remains an urgent healthcare need. OBJECTIVE: In the current study, we investigated a novel class of compounds, the polyisoprenylated cysteinyl amide inhibitors (PCAIs), for their anticancer molecular mechanisms using the NSCLC cell panel with K-Ras and/or other mutant genes. METHODS: The effect of the PCAIs on intracellular K-Ras levels, cell viability, apoptosis, spheroid and colony formation were determined. RESULTS: Treatment of the lung cancer cells with the PCAIs, NSL-RD-035, NSL-BA-036, NSL-BA- 040 and NSL-BA-055 resulted in concentration-dependent cell death in both K-Ras mutant (A549, NCI-H460, and NCI-H1573), N-Ras mutant (NCI-H1299) and other (NCI-H661, NCI-H1975, NCIH1563) NSCLC cells. The PCAIs at 1.0 -10 µM induced the degeneration of 3D spheroid cultures, inhibited clonogenic cell growth and induced marked apoptosis via the extrinsic pathway. The most potent of the PCAIs, NSL-BA-055, at 5 µM induced a seven-fold increase in the activity of caspase- 3/7 and a 75% selective depletion of K-Ras protein levels relative to GAPDH in A549 cells that correlated with PCAIs-induced apoptosis. NSL-BA-040 and NSL-BA-055 also induced the phosphorylation of MAP kinase (ERK 1/2). CONCLUSION: Taken together, PCAIs may be potentially useful as targeted therapies that suppress NSCLC progression through disruption of Ras-mediated growth signaling.

Suppression of focal adhesion formation may account for the suppression of cell migration, invasion and growth of non-small cell lung cancer cells following treatment with polyisoprenylated cysteinyl amide inhibitors.

Migratory cells form extracellular matrix attachments called focal-adhesions. Focal adhesion assembly and disassembly are regulated by the Rho family of small GTPases. We previously reported that polyisoprenylated cysteinyl amide inhibitors (PCAIs) suppress Rho protein levels, disrupting F-actin cytoskeleton remodeling in the formation of lamellipodia and filopodia. In this study, we investigated whether these observations effect focal adhesion formation, which involves cell surface receptors known as integrins and several signaling/adaptor proteins such as vinculin, α-actinin, Rock kinases and phospho-Myosin Light Chain-2 (p-MLC-2), that foster the linkage of the actin cytoskeleton to the extracellular matrix. We observed that treatment of H1299 cells with 5 µM PCAIs for 24 h markedly diminished the level of full-length integrin α4 by at least 24% relative to controls. PCAIs at 5 µM, diminished the levels of vinculin by at least 50%. Immunofluorescent analysis showed at least a 76% decrease in the number of vinculin-focal adhesion punctates. In addition, PCAIs diminished Rock1 levels by 25% and its substrate, p-MLC-2 by 75%. PCAIs did not significantly alter the levels of integrin ß5, α-actinin, and Rock2, suggesting that the effects of the PCAIs are target specific. Our data indicate that the PCAIs alter the levels of the Rho proteins and their effectors to abrogate their functions in cytoskeleton remodeling thereby suppressing focal adhesion formation. This in turn results in a PCAIs-induced decrease in cell invasion, thus making the PCAIs propitious agents for the inhibition of cancer growth and metastasis.

Polyisoprenylated cysteinyl amide inhibitors induce caspase 3/7- and 8-mediated apoptosis and inhibit migration and invasion of metastatic prostate cancer cells.

Metastatic castration-resistant prostate cancer (mCRPC) is the most aggressive and deadly form of prostate cancer. It is characterized by the overexpression of epidermal growth factor receptors whose signals are mediated by small monomeric G proteins of the Ras superfamily. These require polyisoprenylation for functional activity. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) of polyisoprenylated methylated protein methyl esterase (PMPMEase) were developed as potential targeted therapies to mitigate excessive growth signaling in mCRPC either by inhibiting PMPMEase and/or perturbing the polyisoprenylation-dependent functional interactions. We investigated the effects of PCAIs on the viability of prostate cancer PC 3, DU 145, MDA PCa 2b, LNCaP and 22Rv1 cells, determined the effect of the PCAIs on PC 3 cell proliferation, survival and caspase-mediated apoptotic cell death. Metastatic PC 3 and DU 145 cell migration and invasion in the presence of NSL-BA-040 were determined using the scratch and matrigel invasion assays. We further investigated the effect of NSL-BA-040 on F-actin organization in TagRFP F-actin marker-transfected metastatic PC 3 cells. The PCAIs suppress mCRPC cell viability with EC50 values ranging from 1.3 to 4.0 µM for the most potent of the PCAIs against PC 3, DU 145, MDA PCa 2b, LNCaP and 22Rv cells. PCAIs induced apoptotic cell death in PC 3 and DU 145 cells as determined by annexin V/propidium iodide flow cytometry analysis through the activation of caspases 3 and 8 while also inhibiting migration and invasion through the disruption of F-actin organization. Taken together, our studies show the anti-cancer effects on mCRPC cells through induction of caspase-mediated apoptosis and F-actin-mediated inhibition of cell motility and invasion thereby indicating the anti-tumor and anti-metastatic potential of the PCAIs.

Polyisoprenylated cysteinyl amide inhibitors disrupt actin cytoskeleton organization, induce cell rounding and block migration of non-small cell lung cancer.

The malignant potential of Non-Small Cell Lung Cancer (NSCLC) is dependent on cellular processes that promote metastasis. F-actin organization is central to cell migration, invasion, adhesion and angiogenesis, processes involved in metastasis. F-actin remodeling is enhanced by the overexpression and/or hyper-activation of some members of the Rho family of small GTPases. Therefore, agents that mitigate hyperactive Rho proteins may be relevant for controlling metastasis. We previously reported the role of polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential inhibitors of cancers with hyperactive small GTPases. In this report, we investigate the potential role of PCAIs against NSCLC cells and show that as low as 0.5 µM PCAIs significantly inhibit 2D and 3D NCI-H1299 cell migration by 48% and 45%, respectively. PCAIs at 1 µM inhibited 2D and 3D NCI-H1299 cell invasion through Matrigel by 50% and 85%, respectively. Additionally, exposure to 5 µM of the PCAIs for 24 h caused at least a 66% drop in the levels of Rac1, Cdc42, and RhoA and a 38% drop in F-actin intensity at the cell membrane. This drop in F-actin was accompanied by a 73% reduction in the number of filopodia per cell. Interestingly, the polyisoprenyl group of the PCAIs is essential for these effects, as NSL-100, a non-farnesylated analog, does not elicit similar effects on F-actin assembly and organization. Our findings indicate that PCAIs disrupt F-actin assembly and organization to suppress cell motility and invasion. The PCAIs may be an effective therapy option for NSCLC metastasis and invasion control.

Disruption of actin filaments and suppression of pancreatic cancer cell viability and migration following treatment with polyisoprenylated cysteinyl amides.

Pancreatic cancer is characterized by K-Ras mutations in over 90% of the cases. The mutations make the tumors aggressive and resistant to current therapies resulting in very poor prognoses. Valiant efforts to drug mutant K-Ras and related proteins for the treatment of cancers with Ras mutations have been elusive. The need thus persists for therapies to target and suppress the hyperactive K-Ras mutant proteins to normal levels of activity. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) of polyisoprenylated methylated protein methyl esterase (PMPMEase) were designed to disrupt polyisoprenylated protein metabolism and/or functions. The potential for PCAIs to serve as targeted anticancer agents for pancreatic cancer was evaluated in pancreatic ductal adenocarcinoma (PDAC) cell lines expressing mutant (MIAPaCa-2 and Panc-1) and wild type (BxPC-3) K-Ras proteins. The PCAIs inhibited MIAPaCa-2 and BxPC-3 cell viability and induced apoptosis with EC50 values as low as 1.9 µM. The PCAIs, at 0.5 µM, inhibited MIAPaCa-2 cell migration by 50%, inhibited colony formation and disrupted F-actin filament organization. The PCAIs blocked MIAPaCa-2 cell progression at the G0/G1 phase. These results reveal that the PCAIs disrupt pertinent biological processes that lead to pancreatic cancer progression and thus have the potential to act as targeted effective treatments for pancreatic cancer.

The antiangiogenic effects of polyisoprenylated cysteinyl amide inhibitors in HUVEC, chick embryo and zebrafish is dependent on the polyisoprenyl moiety.

Angiogenesis is essential for solid tumor growth, therapeutic resistance and metastasis, the latest accounting for 90% of cancer deaths. Although angiogenesis is essential for the malignant transformations in solid tumors and therefore is an attractive target, few drugs are available that block tumor angiogenesis. The focus has been to block signaling by receptor tyrosine kinases (RTKs), such as for vascular endothelial growth factor (VEGF), whose activation abrogate apoptosis and promote angiogenesis. The polyisoprenylated cysteinyl amide inhibitors (PCAIs) were designed to modulate aberrant polyisoprenylated small G-proteins such as mutant Ras whose constitutive activation promotes RTKs signaling. Since polyisoprenylation is essential for protein-protein interactions and functions of G-proteins, we hypothesized that the PCAIs would disrupt the monomeric G-protein signaling thereby effectively inhibiting angiogenesis. In this study we determined the effects of PCAIs on human umbilical vein endothelial cells (HUVEC) tube formation, cell viability, cell migration and invasion as well as in vivo using the chick chorioallantoic membrane (CAM) and zebrafish models. At sub- to low micromolar concentrations, the PCAIs inhibit the native and VEGF-stimulated cell migration and invasion as well as tube formation and angiogenesis in CAM and zebrafish embryos. The concentrations that block the angiogenic processes were lower than those that induce cell death. Since angiogenesis is essential for tumor growth but otherwise limited to wound healing, feeding fat cells and uterine wall repair in adults, it is conceivable that these compounds can be developed into safer therapeutics for cancers and retinal neovascularization that leads to loss of vision.

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.

Further evaluation of the tropane analogs of haloperidol.

Previous work from our labs has indicated that a tropane analog of haloperidol with potent D2 binding but designed to avoid the formation of MPP(+)-like metabolites, such as 4-(4-chlorophenyl)-1-(4-(4-fluorophenyl)-4-oxobutyl)pyridin-1-ium (BCPP(+)) still produced catalepsy, suggesting a strong role for the D2 receptor in the production of catalepsy in rats, and hence EPS in humans. This study tested the hypothesis that further modifications of the tropane analog to produce compounds with less potent binding to the D2 receptor than haloperidol, would produce less catalepsy. These tests have now revealed that while haloperidol produced maximum catalepsy, these compounds produced moderate to low levels of catalepsy. Compound 9, with the least binding affinity to the D2R, produced the least catalepsy and highest Minimum Adverse Effective Dose (MAED) of the analogs tested regardless of their affinities at other receptors including the 5-HT1AR. These observations support the hypothesis that moderation of the D2 binding of the tropane analogs could reduce catalepsy potential in rats and consequently EPS in man.

Polyisoprenylated methylated protein methyl esterase: a putative biomarker and therapeutic target for pancreatic cancer.

Pancreatic cancer is the most deadly neoplasm with a 5-year survival rate of less than 6%. Over 90% of cases harbor K-Ras mutations, which are the most challenging to treat due to lack of effective therapies. Here, we reveal that polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed in 93% of pancreatic ductal adenocarcinoma. We further present polyisoprenylated cysteinyl amide inhibitors (PCAIs) as novel compounds designed with structural elements for optimal in vivo activities and selective disruption of polyisoprenylation-mediated protein functions. 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 and farnesylthiosalicylamide were ineffective at 20 µM. The PCAIs thus have the potential to serve as effective therapies for pancreatic and other cancers with hyperactive growth signaling pathways mediated by Ras and related G-proteins.

Identification of a new selective dopamine D4 receptor ligand.

The dopamine D4 receptor has been shown to play key roles in certain CNS pathologies including addiction to cigarette smoking. Thus, selective D4 ligands may be useful in treating some of these conditions. Previous studies in our laboratory have indicated that the piperazine analog of haloperidol exhibits selective and increased affinity to the DAD4 receptor subtype, in comparison to its piperidine analog. This led to further exploration of the piperazine moiety to identify new agents that are selective at the D4 receptor. Compound 27 (KiD4=0.84 nM) was the most potent of the compounds tested. However, it only had moderate selectivity for the D4 receptor. Compound 28 (KiD4=3.9 nM) while not as potent, was more discriminatory for the D4 receptor subtype. In fact, compound 28 has little or no binding affinity to any of the other four DA receptor subtypes. In addition, of the 23 CNS receptors evaluated, only two, 5HT1AR and 5HT2BR, have binding affinity constants better than 100 nM (Ki <100 nM). Compound 28 is a potentially useful D4-selective ligand for probing disease treatments involving the D4 receptor, such as assisting smoking cessation, reversing cognitive deficits in schizophrenia and treating erectile dysfunction. Thus, further optimization, functional characterization and evaluation in animal models may be warranted.

Polyisoprenylated methylated protein methyl esterase overexpression and hyperactivity promotes lung cancer progression.

The involvement of hyperactive polyisoprenylated proteins in cancers has stimulated the search for drugs to target and suppress their excessive activities. Polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition has been shown to modulate polyisoprenylated protein function. For PMPMEase inhibition to be effective against cancers, polyisoprenylated proteins, the signaling pathways they mediate and/or PMPMEase must be overexpressed, hyperactive and be involved in at least some cases of cancer. PMPMEase activity in lung cancer cells and its expression in lung cancer cells and cancer tissues were investigated. PMPMEase was found to be overexpressed and significantly more active in lung cancer A549 and H460 cells than in normal lung fibroblasts. In a tissue microarray study, PMPMEase immunoreactivity was found to be significantly higher in lung cancer tissues compared to the normal controls (p < 0.0001). The mean scores ± SEM were 118.8 ± 7.7 (normal), 232.1 ± 25.1 (small-cell lung carcinomas), 352.1 ± 9.4 (squamous cell carcinomas), 311.7 ± 9.8 (adenocarcinomas), 350.0 ± 24.2 (papillary adenocarcinomas), 334.7 ± 30.1 (adenosquamous carcinomas), 321.9 ± 39.7 (bronchioloalveolar carcinomas), and 331.3 ± 85.0 (large-cell carcinomas). Treatment of lung cancer cells with L-28, a specific PMPMEase inhibitor, resulted in concentration-dependent cell death (EC50 of 8.5 µM for A549 and 2.8 µM for H460 cells). PMPMEase inhibition disrupted actin filament assembly, significantly inhibited cell migration and altered the transcription of cancer-related genes. These results indicate that elevated PMPMEase activity spur cell growth and migration, implying the possible use of PMPMEase as a protein biomarker and drug target for lung cancer.

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.