Discovery of a Novel Class of d-Amino Acid Oxidase Inhibitors Using the Schrödinger Computational Platform.

d-Serine is a coagonist of the N-methyl d-aspartate (NMDA) receptor, a key excitatory neurotransmitter receptor. In the brain, d-serine is synthesized from its l-isomer by serine racemase and is metabolized by the D-amino acid oxidase (DAO, DAAO). Many studies have linked decreased d-serine concentration and/or increased DAO expression and enzyme activity to NMDA dysfunction and schizophrenia. Thus, it is feasible to employ DAO inhibitors for the treatment of schizophrenia and other indications. Powered by the Schrödinger computational modeling platform, we initiated a research program to identify novel DAO inhibitors with the best-in-class properties. The program execution leveraged an hDAO FEP+ model to prospectively predict compound potency. A new class of DAO inhibitors with desirable properties has been discovered from this endeavor. Our modeling technology on this program has not only enhanced the efficiency of structure-activity relationship development but also helped to identify a previously unexplored subpocket for further optimization.

FKBPL and its peptide derivatives inhibit endocrine therapy resistant cancer stem cells and breast cancer metastasis by downregulating DLL4 and Notch4.

BACKGROUND: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. METHODS: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test. RESULTS: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4. CONCLUSION: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

An evaluation of the consequences of using short measures of the Big Five personality traits.

Researchers often use very abbreviated (e.g., 1-item, 2-item) measures of personality traits due to their convenience and ease of use as well as the belief that such measures can adequately capture an individual's personality. Using data from 2 samples (N = 437 employees, N = 355 college students), we show that this practice, particularly the use of single-item measures, can lead researchers to substantially underestimate the role that personality traits play in influencing important behaviors and thereby overestimate the role played by new constructs. That is, the use of very short measures of personality may substantially increase both the Type 1 and Type 2 error rates. We argue that even slightly longer measures can substantially increase the validity of research findings without significant inconvenience to the researcher or research participants.

FKBPL and peptide derivatives: novel biological agents that inhibit angiogenesis by a CD44-dependent mechanism.

PURPOSE: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. EXPERIMENTAL DESIGN: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. RESULTS: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. CONCLUSION: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.

FKBPL regulates estrogen receptor signaling and determines response to endocrine therapy.

The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor alpha (ERalpha) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERalpha phosphorylation on Ser(118) in response to 17beta-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.

A novel FK506-like binding protein interacts with the glucocorticoid receptor and regulates steroid receptor signaling.

FKBP-like (FKBPL) protein is a novel immunophilin-like protein that plays a role in the cellular stress response. Its three tetratricopeptide repeat motifs are homologous to the heat shock protein 90 interaction sites of other immunophilins that have roles in steroid hormone receptor signaling. In this study, using biomolecular complementation and coimmunoprecipitation techniques, we show that FKBPL also colocalizes and interacts with the components of the heat shock protein 90-glucocorticoid receptor (GR) complex and demonstrate that the PPIase domain of FKBPL is important for the interaction between this complex and the dynein motor protein, dynamitin. Treatment of DU145 cells with the GR ligand, dexamethasone, induced a rapid and coordinated translocation of both GR and FKBPL to the nucleus; this response was perturbed when FKBPL was knocked down with a targeted small interfering RNA. Furthermore, overexpression of FKBPL increased GR protein levels and transactivation of a luciferase reporter gene in response to dexamethasone in DU145 cells. However, these responses were cell line dependent. In summary, these data suggest that FKBPL can be classed as a new member of the FKBP protein family with a role in steroid receptor complexes and signaling.

Evaluation of the antiangiogenic potential of AQ4N.

PURPOSE: A number of cytotoxic chemotherapy agents tested at low concentrations show antiangiogenic properties with limited cytotoxicity, e.g., cyclophosphamide, tirapazamine, and mitoxantrone. AQ4N is a bioreductive alkylaminoanthraquinone that is cytotoxic when reduced to AQ4; hence, it can be used to target hypoxic tumor cells. AQ4N is structurally similar to mitoxantrone and was evaluated for antiangiogenic properties without the need for bioreduction. EXPERIMENTAL DESIGN: The effect of AQ4N and fumagillin on human microvascular endothelial cells (HMEC-1) was measured using a variety of in vitro assays, i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound scrape, tubule formation, rat aortic ring, and invasion assays. Low-dose AQ4N (20 mg/kg) was also given in vivo to mice bearing a tumor in a dorsal skin flap. RESULTS: AQ4N (10(-11) to 10(-5) mol/L) had no effect on HMEC-1 viability. AQ4N (10(-9) to 10(-5)mol/L) caused a sigmoidal dose-dependent inhibition of endothelial cell migration in the wound scrape model. Fumagillin showed a similar response over a lower dose range (10(-13) to 10(-9) mol/L); however, the maximal inhibition was less (25% versus 43% for AQ4N). AQ4N inhibited HMEC-1 cell contacts on Matrigel (10(-8) to 10(-5) mol/L), HMEC-1 cell invasion, and sprouting in rat aorta explants. Immunofluorescence staining with tubulin, vimentim, dynein, and phalloidin revealed that AQ4N caused disruption to the cell cytoskeleton. When AQ4N (20 mg/kg) was given in vivo for 5 days, microvessels disappeared in LNCaP tumors grown in a dorsal skin flap. CONCLUSIONS: This combination of assays has shown that AQ4N possesses antiangiogenic effects in normoxic conditions, which could potentially contribute to antitumor activity.