Tryphon Mazu

δ-Carbolines and their ring-opened analogs: Synthesis and evaluation against fungal and bacterial opportunistic pathogens

Previous studies have indicated that the δ-carboline (2) ring system derived from the natural product cryptolepine (1) may represent a pharmacophore for anti-infective activity. This paper describes the design and synthesis of a small library of substituted δ-carbolines and the evaluation of the anti-fungal and anti-bacterial activities. An evaluation of the anti-bacterial activity of a previously reported library of ring-opened analogs was also conducted to provide an opportunity to test the hypothesis that both group of compounds may have the same biological target. Results indicate that against a selected group of fungal pathogens, substituted δ-carbolinium analogs displayed higher potency and several fold lower cytotoxicity than cryptolepine the parent natural product. Both the δ-carbolinium compounds and their ring-opened analogs, exhibited equally high anti-bacterial activity against the selected pathogens and especially against the gram positive bacteria evaluated.

The Mechanistic Targets of Antifungal Agents: An Overview

Pathogenic fungi are a major causative group for opportunistic infections (OIs). AIDS patients and other immunocompromised individuals are at risk for OIs, which if not treated appropriately, contribute to the mortality associated with their conditions. Several studies have indicated that the majority of HIV-positive patients contract fungal infections throughout the course of their disease. Similar observations have been made regarding the increased frequency of bone marrow and organ transplants, the use of antineoplastic agents, the excessive use of antibiotics, and the prolonged use of corticosteroids among others. In addition, several pathogenic fungi have developed resistance to current drugs. Together these have conspired to spur a need for developing new treatment options for OIs. To aid this effort, this article reviews the biological targets of current and emerging drugs and agents that act through these targets for the treatment of opportunistic fungal infections.

Identification of 3-phenylaminoquinolinium and 3-phenylaminopyridinium salts as new agents against opportunistic fungal pathogens

Previous studies on the indoloquinoline alkaloid, cryptolepine (2), revealed that it has antii-nfective properties among other activities. Using Structure-activity relationship (SAR) techniques, several ring-opened analogs of cryptolepine (3-phenylaminopyridinium and 3-phenylaminoquinolinium derivatives) were designed to improve the potency and lower the cytotoxicity shown by several of the precursor agents. Results indicate that these ring-opened analogs constitute new anti-infective agents with over a 100-fold potency and several fold lower cytotoxicity than cryptolepine from which they are derived.

Inactivation of chromatin remodeling factors sensitizes cells to selective cytotoxic stress

The SWI/SNF chromatin-remodeling complex plays an essential role in several cellular processes including cell proliferation, differentiation, and DNA repair. Loss of normal function of the SWI/SNF complex because of mutations in its subunits correlates with tumorigenesis in humans. For many of these cancers, cytotoxic chemotherapy is the primary, and sometimes the only, therapeutic alternative. Among the antineoplastic agents, anthracyclines are a common treatment option. Although effective, resistance to these agents usually develops and serious dose-related toxicity, namely, chronic cardiotoxicity, limits its use. Previous work from our laboratory showed that a deletion of the SWI/SNF factor SNF2 resulted in hypersensitivity to doxorubicin. We further investigated the contribution of other chromatin remodeling complex components in the response to cytotoxic chemotherapy. Our results indicate that, of the eight SWI/SNF strains tested, snf2, taf14, and swi3 were the most sensitive and displayed distinct sensitivity to different cytotoxic agents, while snf5 displayed resistance. Our experimental results indicate that the SWI/SNF complex plays a critical role in protecting cells from exposure to cytotoxic chemotherapy and other cytotoxic agents. Our findings may prove useful in the development of a strategy aimed at targeting these genes to provide an alternative by hypersensitizing cancer cells to chemotherapeutic agents.

Defects in Base Excision Repair Sensitize Cells to Manganese in S. cerevisiae

Manganese (Mn) is essential for normal physiologic functioning; therefore, deficiencies and excess intake of manganese can result in disease. In humans, prolonged exposure to manganese causes neurotoxicity characterized by Parkinson-like symptoms. Mn2+ has been shown to mediate DNA damage possibly through the generation of reactive oxygen species. In a recent publication, we showed that Mn induced oxidative DNA damage and caused lesions in thymines. This study further investigates the mechanisms by which cells process Mn2+-mediated DNA damage using the yeast S. cerevisiae. The strains most sensitive to Mn2+ were those defective in base excision repair, glutathione synthesis, and superoxide dismutase mutants. Mn2+ caused a dose-dependent increase in the accumulation of mutations using the CAN1 and lys2-10A mutator assays. The spectrum of CAN1 mutants indicates that exposure to Mn results in accumulation of base substitutions and frameshift mutations. The sensitivity of cells to Mn2+ as well as its mutagenic effect was reduced by N-acetylcysteine, glutathione, and Mg2+. These data suggest that Mn2+ causes oxidative DNA damage that requires base excision repair for processing and that Mn interferes with polymerase fidelity. The status of base excision repair may provide a biomarker for the sensitivity of individuals to manganese.

Abstract 2545: Effect of aldehydes in anthracycline cytotoxicity in S. cerevisiae

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Anthracyclines constitute drugs of choice as single agents or in combination therapy in cancers that are refractory to other therapies, such as triple negative breast cancer, commonly observed in African-American women (AA) and is characterized for lacking biomarkers for targeted therapy, and for being aggressive and carrying bad prognoses. Anthracyclines effectiveness is dose-dependent, which increases the risk of side-effects and eventually the development of drug resistance. In S. cerevisiae, mutants of HOM6 display hypersensitivity to doxorubicin by inactivation of the homoserine dehydrogenase. We hypothesized that the L-aspartate-semialdehyde accumulating at this step, sensitizes the cells to anthracyclines. We confirmed this by inactivating HOM3, a prior step in the threonine biosynthesis pathway which prevents the accumulation of the L-aspartate-semialdehyde, rescuing the hom6 strain. To expand our knowledge of the relationship between aldehydes and anthracyclines, we sought to identify other aldehydes that can enhance the toxic effects of doxorubicin. We exposed S. cerevisiae to aldehydes (acetaldehyde, formaldehyde and glutaraldehyde) alone and combined with doxorubicin, cisplatin and menadione. Combination of formaldehyde (2 mM) and doxorubicin was most effective at reducing cell survival by between 31 to 39-fold (in wild type cells) relative to doxorubicin and formaldehyde alone. This effect was dose-dependent on doxorubicin. Co-treatment with formaldehyde and doxorubicin also showed increased toxicity in anthracycline-resistant strains siz1 and msh2. Combination of formaldehyde (2 mM) and menadione (60 mM) reduced cell survival by between 1- to 1.5-fold in wild type cells. Combination of acetaldehyde (2 mM) and menadione (60 mM) reduced cell survival by between 1.6- to 2.2-fold in wild type cells. Wild type cells treated with the combination of cisplatin (80 μM) and acetaldehyde (2 mM) or glutaraldehyde (2 mM) showed a reduced cell survival by between 0.4- to 3.12-fold with the combination of cisplatin and glutaraldehyde being the more cytotoxic of the two. Acetaldehyde, formaldehyde and glutaraldehyde show enhanced toxicity of wild type cells when combined with cytotoxic drugs. The potential use of a combination of aldehydes and cytotoxic drugs could potentially lead to applications intended to enhance anthracycline-based therapy. Citation Format: Jana S. Miles, Tryphon K. Mazu, Hernan Flores-Rozas. Effect of aldehydes in anthracycline cytotoxicity in S. cerevisiae. [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 2545. doi:10.1158/1538-7445.AM2015-2545

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

Abstract 1785: Distinct effect of aldehydes in anthracycline cytotoxicity in S. cerevisiae

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Anthracyclines are common chemotherapeutic agents used in many cancers which are refractory to other therapeutic alternatives. In triple negative breast cancer which is commonly observed in African-American women (AA) and tend to be more aggressive, carry worse prognoses and are harder to manage because they lack the molecular targets required for therapies, anthracyclines are some of the few therapeutic alternatives. Although effective, resistance to this agent usually arises and serious side-effects like the development of cardiomyopathy limits their use. To increase our knowledge of the pathways that protect cells from anthracyclines we carried out a genome-wide genetic screening in the eukaryotic model organism S. cerevisiae to identify gene defects that generate cells hypersensitive to doxorubicin. A total of 71 deletion strains displayed varying levels of sensitivity to anthracyclines. Among these, mutants of HOM6 confer significant increased sensitivity to doxorubicin. This effect appears to be due to the accumulation of the threonine intermediate L-aspartate-semialdehyde and can be abolished by the inactivation of a prior step, involving HOM3, which prevents the accumulation of the intermediate. In order to further characterize the synergism of aldehydes and doxorubicin, we have tested the effect of different aldehydes on the cytotoxicity of cells exposed to anthracyclines. Our data show that treatment with formaldehyde (2 mM) reduced the survival of the wild type strain to 78%. However, in the presence of doxorubicin, formaldehyde reduced the viability of the strain by between 31 to 39-fold (with 10 µM doxorubicin) relative to doxorubicin and formaldehyde alone and by ∼110-fold relative to doxorubicin at 150 µM concentration. Co-treatment with formaldehyde and doxorubicin of anthracycline-resistant strains siz1 and msh2 also shows increased toxicity. However, the synergistic effect is not as pronounced in these strains compared to the wild type. Among the aldehydes tested, formaldehyde was most effective. Glutaraldehyde and acetaldehyde displayed distinct effect on strains siz1, and ald5. The potential use of a combination of aldehydes and cytotoxic drugs could potentially lead to applications intended to enhance anthracycline-based therapy. This possibility will be discussed, as well as the targeting of aldehydes in relevant human pathways. Research supported by NIH/NCRR RCMI grant #RR03020 at Florida A&M University. Citation Format: Jana Sharrae Miles, Tryphon Mazu, Selina Darling-Reed, Hernan Flores-Rozas. Distinct effect of aldehydes in anthracycline cytotoxicity in S. cerevisiae. [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 1785. doi:10.1158/1538-7445.AM2014-1785

Abstract C50: Identification of secondary targets to enhance anthracycline-based therapy

Breast cancer is the most common cancer in women worldwide. African-American women (AA) tend to have more aggressive breast cancers that present more frequently as estrogen receptor, progesterone receptor (PR) and HER2 negative which are referred to as “triple negative” breast cancers (TNBC). TNBC tumors carry worse prognoses and are harder to manage because they lack the molecular targets required for therapies. In patients suffering from TNBC cytotoxic agents such as anthracyclines are some of the few therapeutic alternatives. Anthracyclines are among the most common chemotherapeutic agents used to-date. Although effective, resistance to this agent usually develops in the course of treatment and serious dose-related toxicity, namely chronic cardio-toxicity, limits their use. To increase our knowledge of the pathways that protect cells from anthracyclines we carried out a genome-wide genetic screening in the eukaryotic model organism S. cerevisiae to identify gene defects that generate cells hypersensitive to doxorubicin. A total of 71 deletion strains displayed varying levels of sensitivity to anthracyclines. Major pathways were represented by multiple genes, including homologous recombination/DNA repair, the heat-shock response, chromatin remodeling and aminoacid metabolism. To further investigate the mechanisms that render cells hypersensitive to anthracyclines when genes of these pathways are inactivated, we determined the sensitivity of several mutants to a series of cytotoxic agents. Our findings indicate that inactivation of the heat-shock protein 40 encoded by the genes YDJ1 and ZUO1, as well as the heat-shock protein 70 encoded by the gene SSZ1, render cells highly sensitive to the toxicity of doxorubicin and cisplatin. In addition, deletion of chromatin remodeling factor SNF2 resulted in sensitivity to doxorubicin of 100,000-fold higher than wild type cells and it is also sensitive to daunorubicin, cisplatin, as well as to DNA alkylating agents, topoI inhibitor campthotecin or replication blocks from hydroxyurea. Interestingly, mutations of the HOM6 gene increases the accumulation of an aldehyde intermediate that synergizes with anthracyclines. Taken together, our data indicates that inactivation of any of these pathways can be targeted to hypersensitize cancer cells to cytotoxic agents to enhance chemotherapeutic efficacy in TNBC. This project was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 2 G12 RR003020 and 8 G12 MD007582-28. Citation Format: Hernan Flores-Rozas, Tryphon Mazu, Miles D. Freeman, Jana S. Miles. Identification of secondary targets to enhance anthracycline-based therapy. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C50. doi:10.1158/1538-7755.DISP13-C50

Abstract 4367: Differential contribution of chromatin remodeling complex components to the protection from cytotoxic stress.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Anthracyclines are among the most common chemotherapeutic agents used to-date. Although effective, resistance to this agent usually develops in the course of treatment and serious dose-related toxicity, namely chronic cardio-toxicity, limits its use. In patients suffering from triple negative breast cancer (TNBC) cytotoxic agents such as anthracyclines are some of the only therapeutic alternatives. To increase our knowledge of the pathways that protect cells from anthracyclines we carried out a genome-wide genetic screening in the eukaryotic model organism S. cerevisiae to identify gene defects that generate cells hypersensitive to doxorubicin. A total of 71 deletion strains displayed varying levels of sensitivity to anthracylcines. Of these, 11 genes are involved in chromatin remodeling. The most sensitive mutations corresponded to swi3, snf2 and arp8, which are components of the SWI/SNF complex. Deletion of SNF2 resulted in sensitivity to doxorubicin of 100,000-fold higher than wild type cells. Further analysis of the SNF2 defective strain revealed that it is also sensitive to daunorubicin and cisplatin, as well as to other DNA damaging agents such as alkylating agents, the topoI inhibitor campthotecin or replication blocks from hydroxyurea. Our results indicate that the SWI/SNF complex plays a critical role in protecting cells from exposure to anthracyclines and other DNA damaging agents. Targeting these genes may provide a useful alternative to hypersensitize cancer cells to chemotherapy and provide a therapeutic alternative to TNBC. Research supported by NIH/NCRR RCMI grant #RR03020 at Florida A&M University. Citation Format: Miles D. Freeman, Tryphon Mazu, Hernan Flores-Rozas. Differential contribution of chromatin remodeling complex components to the protection from cytotoxic stress. [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 4367. doi:10.1158/1538-7445.AM2013-4367