Cynthia B. Myers

An Isoform-Selective, Small-Molecule Inhibitor Targets the Autoregulatory Mechanism of p21-Activated Kinase

Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, preactivated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate an alternative strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak.

Influence of the Length and Positioning of the Antiestrogenic Side Chain of Endoxifen and 4-Hydroxytamoxifen on Gene Activation and Growth of Estrogen Receptor Positive Cancer Cells

Tamoxifen has biologically active metabolites: 4-hydroxytamoxifen (4OHT) and endoxifen. The E-isomers are not stable in solution as Z-isomerization occurs. We have synthesized fixed ring (FR) analogues of 4OHT and endoxifen as well as FR E and Z isomers with methoxy and ethoxy side chains. Pharmacologic properties were documented in the MCF-7 cell line, and prolactin synthesis was assessed in GH3 rat pituitary tumor cells. The FR Z-isomers of 4OHT and endoxifen were equivalent to 4OHT and endoxifen. Other test compounds used possessed partial estrogenic activity. The E-isomers of FR 4OHT and endoxifen had no estrogenic activity at therapeutic serum concentrations. None of the newly synthesized compounds were able to down-regulate ER levels. Molecular modeling demonstrated that some compounds would each create a best fit with a novel agonist conformation of the ER. The results demonstrate modulation by the ER complex of cell replication or gene transcription in cancer.

Adenosine-5′-O-(2-fluorodiphosphate) (ADPβF), an analog of adenosine-5′-phosphosulfate

Abstract Adenosine-5′-phosphosulfate (APS) and its 3′-phosphorylated metabolite PAPS (3′-phosphoadenosine-5′-phosphosulfate) play central roles in biological systems. Few analogs of APS or PAPS have been described, partially due to the instability of the phosphosulfate moiety. ADPβF, adenosine-5′-O-(2-fluorodiphosphate), an ADP derivative in which one of the nonbridge oxygens on the terminal phosphoryl group is replaced by fluorine, has been synthesized by reaction of AMP-morpholidate with bis(tributylammonium)fluorophosphate. In contrast to APS, ADPβF is stable at room temperature for at least a day from pH 3 to 10. ADPβF is an alternate substrate for adenosine phosphosulfate kinase (ATP:APS 3′-phosphotransferase) yielding the PAPS analog PADPβF. In the APS kinase reaction ADPβF has a Km 7-fold greater than that of APS, and a Vmax that is 9% that of APS. ADPβF is an alternate substrate for the reverse reaction of ATP sulfurylase (ATP:sulfate adenylyltransferase), yielding ATP and FPO3 as products; ADPβF has a Vmax that is 12% of the Vmax with APS and a 1.4-fold higher Km value than APS. ADPβF can conveniently be synthesized from ATP and FPO3 in the presence of catalytic amounts of ATP sulfurylase and inorganic pyrophosphatase. ADPβF is a poor ADP analog for two enzymes tested: high concentrations did not inhibit pyruvate kinase or hexokinase, and ADPβF did not replace ADP in the reverse pyruvate kinase reaction. ADPβF, like APS, has one less charge than ADP at neutral pH. These results suggest that a major determinant in enzymatic discrimination between APS and ADP is the charge difference between the terminal sulfuryl and phosphoryl groups.

Chemical Genetic Screening for Compounds That Preferentially Inhibit Growth of Methylthioadenosine Phosphorylase (MTAP)–Deficient Saccharomyces cerevisiae

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP-deficient cells. To determine if MTAP inactivation could be targeted, the authors have performed a differential chemical genetic screen in isogenic MTAP+ and MTAP− Saccharomyces cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP− yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP− cells exhibiting a 4-fold shift in IC50 compared to MTAP+ cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP− yeast. These compounds were also examined for growth inhibition of isogenic MTAP+ and MTAP− HT1080 fibrosarcoma cells, and 4 of the 5 compounds exhibited evidence of modest but significant increased potency in MTAP− cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP− cells.

The Effect of 5-Aminolevulinic Acid and Its Derivatives on Protoporphyrin IX Accumulation and Apoptotic Cell Death in Castrate-resistant Prostate Cancer Cells

OBJECTIVE To examine whether pharmacologically relevant zinc-binding agents are capable of depleting X-linked inhibitor of apoptosis protein in tumor cells. Our prior work reveals that treatment with zinc-chelating agents induces selective downregulation of the X-linked inhibitor of apoptosis protein in cancer cells of various origins. A precursor of the heme synthetic pathway, 5-aminolevulinic acid, is metabolized to protoporphyrin IX, which is highly reactive with zinc. We assessed whether modified versions of 5-aminolevulinic acid with lipophilic side chains can enhance efficacy and selectivity with respect to protoporphyrin IX accumulation, X-linked inhibitor of apoptosis protein depletion, and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human castration-resistant prostate cancer cells. METHODS Seven modified versions of 5-aminolevulinic acid (5 esters and 2 amides) were synthesized. Levels of endogenous protoporphyrin IX were examined by flow cytometry. X-linked inhibitor of apoptosis protein expression was examined by Western blotting. terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay was used to assess cell apoptosis. Results were compared qualitatively. RESULTS Accumulation of endogenous protoporphyrin IX by castration-resistant prostate cancer cells was shown to be directly related to the carbon chain length of the esterified 5-aminolevulinic acid derivatives. In fact, treatment with 5-aminolevulinic acid-HE was superior to that achieved by 5-aminolevulinic acid with respect to X-linked inhibitor of apoptosis protein downregulation. 5-aminolevulinic acid and 5-aminolevulinic acid-HE in combination with tumor necrosis factor-related apoptosis-inducing ligand significantly enhanced apoptotic cell death in castration-resistant prostate cancer cell lines. CONCLUSION Esterified derivatives of 5-aminolevulinic acid alone or in combination with other agents may provide therapeutic opportunities in the treatment of castration-resistant prostate cancer by harnessing apoptotic pathways that are triggered by cellular zinc imbalance.

Synthesis and X-ray crystal structure of 3-deoxy-3-fluoro-β-D-allopyranoside

The synthesis of a novel compound, 3-deoxy-3-fluoro-β-d-allopyranoside, is described. It involves blocking the 4- and 6-positions of allose to cause fluorination only at the 3-position. This six-step synthesis gave pure crystalline compound in a 5% overall yield. The crystal structure, determined by X-ray diffraction, confirmed the chemical formula and configuration of the product. It showed that the C-F bond isaxial, while the C-(OH) bonds areequatorial. The overall conformation of this fluoroallose is similar to that of allose (Acta Cryst.1984,C40, 1863) (with OH instead of F), but the packing in the two crystals is different in the region of C3, the site of chemical variation.

Abstract 4409: The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tamoxifen is the most widely used agent in the world for antihormonal therapy of Estrogen Receptor (ER)-positive breast cancer for both pre- and postmenopausal patients. Tamoxifen is a pro-drug and needs to be metabolically activated to a more potent antiestrogen with a higher affinity for the ER. Initially 4-hydroxytamoxifen (4OHT) was the active metabolite of interest, but more recently 4-hydroxy-N-desmethyltamoxifen (endoxifen) is also of significant interest. The role of endoxifen in therapy remains controversial, however both 4OHT and endoxifen exist in two isomeric forms- cis and trans. Previously, it was shown that cis-isomers of tamoxifen metabolites have weak anti-estrogenic properties using in vitro models available (Jordan VC et al, Endocrinology, 1987, 122(4); 1449-54). However, studies show that an increase of cis-isomers of 4OHT in animals and patients may contribute to tamoxifen resistance (Osborne CK et al, JNCI, 1991, 83; 1477-82; Osborne CK et al, J Clin Oncol, 1992, 10; 304-10). Thus, it is important to know the estrogenic properties of clinically relevant tamoxifen metabolites. To document the anti-estrogenic profiles of both geometric isomers of two major hydroxylated metabolites of tamoxifen, isomerically stable fixed-ring derivatives of 4OHT and endoxifen were synthesized. We have assessed the anti-estrogenic properties of these compounds using cell proliferation assays in selectively cloned hypersensitive ER-positive human breast cancer cell lines MCF-7:WS8 and T47D:A18. Also to assess to estrogenic/anti-estrogenic properties of synthesized compounds on transcriptional level, we have used real-time PCR of rat Prolactin (Prl) mRNA isolated from GH3 rat pituitary cell line. Our results indicate that the fixed-ring trans-4OHT and trans-Endoxifen are potent antiestrogens and equivalent to each other, and to their non-fixed-ring counterparts in both cell proliferation assays and Prl gene activity, as they do not possess any agonistic activities by themselves and are able to inhibit the estrogenic actions of 0,1nM 17β-estradiol (E2) completely (IC50 1nM). However, cis-isomers of the fixed-ring compounds are very weakly estrogenic by themselves but are very weak anti-estrogens in combination with 0,1nM E2 in all assays. Conclusions: the weak estrogen-like activity and low potency of the cis-isomers is unlikely to influence the growth of breast cancer during long-term therapy. The high potency of trans-endoxifen and 4OHT will dominate the control of the tumor growth. Support: This study (PYM) was supported by Susan G. Komen for the Cure International Postdoctoral Fellowship under award number SAC100009 (VCJ). Citation Format: Philipp Y. Maximov, Cynthia Myers, Daphne J. Fernandes, V. Craig Jordan. The characterization of isomerically stable fixed ring derivatives of tamoxifen metabolites 4-hydroxytamoxifen and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in vitro. [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 4409. doi:10.1158/1538-7445.AM2013-4409