Sonia Martinez

A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations

Oncogene activation has been shown to generate replication-born DNA damage, also known as replicative stress. The primary responder to replicative stress is not Ataxia-Telangiectasia Mutated (ATM) but rather the kinase ATM and Rad3-related (ATR). One limitation for the study of ATR is the lack of potent inhibitors. We here describe a cell-based screening strategy that has allowed us to identify compounds with ATR inhibitory activity in the nanomolar range. Pharmacological inhibition of ATR generates replicative stress, leading to chromosomal breakage in the presence of conditions that stall replication forks. Moreover, ATR inhibition is particularly toxic for p53-deficient cells, this toxicity being exacerbated by replicative stress–generating conditions such as the overexpression of cyclin E. Notably, one of the compounds we identified is NVP-BEZ235, a dual phosphatidylinositol-3-OH kinase (PI3K) and mTOR inhibitor that is being tested for cancer chemotherapy but that we now show is also very potent against ATM, ATR and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).

Pten Positively Regulates Brown Adipose Function, Energy Expenditure, and Longevity

Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Pten(tg) mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Pten(tg) mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Pten(tg) mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Pten(tg) fibroblasts programmed with Prdm16 and Cebpβ form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.

Pharmacological Inhibition of PI3K Reduces Adiposity and Metabolic Syndrome in Obese Mice and Rhesus Monkeys

Genetic inhibition of PI3K signaling increases energy expenditure, protects from obesity and metabolic syndrome, and extends longevity. Here, we show that two pharmacological inhibitors of PI3K, CNIO-PI3Ki and GDC-0941, decrease the adiposity of obese mice without affecting their lean mass. Long-term treatment of obese mice with low doses of CNIO-PI3Ki reduces body weight until reaching a balance that is stable for months as long as the treatment continues. CNIO-PI3Ki treatment also ameliorates liver steatosis and decreases glucose serum levels. The above observations have been recapitulated in independent laboratories and using different oral formulations of CNIO-PI3Ki. Finally, daily oral treatment of obese rhesus monkeys for 3 months with low doses of CNIO-PI3Ki decreased their adiposity and lowered their serum glucose levels, in the absence of detectable toxicities. Therefore, pharmacological inhibition of PI3K is an effective and safe anti-obesity intervention that could reverse the negative effects of metabolic syndrome in humans.

Novel Approach for Chemotype Hopping Based on Annotated Databases of Chemically Feasible Fragments and a Prospective Case Study: New Melanin Concentrating Hormone Antagonists

A novel strategy for chemotype hopping, based on annotated databases of chemically feasible fragments and their oriented functionalization, is presented. A three-dimensional (3D) similarity analysis of project-oriented functionalized scaffolds provides a prioritized proposal for synthesis with the most appropriate linkers and optimal regiochemistry on R-groups. This strategy maximizes the potential of proprietary and commercially available compounds. A retrospective and prospective case study, on melanin concentrating hormone (MCH) antagonists, showing the impact on the drug discovery process of this new strategy by maintaining primary activity and improving key ADME/Tox property while enhancing intellectual property (IP) position is demonstrated.

Synthesis of 3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles, displaying combined 5-HT uptake inhibiting and α2-adrenoceptor antagonistic activities: a novel series of potential antidepressants

The synthesis of a series of novel 3-piperazinylmethyl-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles as novel dual 5-HT reuptake inhibitors and alpha(2)-adrenoceptor antagonists is described. Their affinity at the three different human alpha(2)-adrenoceptor subtypes and the 5-HT transporter site is reported. The in vivo activity of the compounds was measured in two different assays: (1). inhibition of pCA-induced excitation, which evaluates the ability to block the central 5-HT transporter, and (2). inhibition of xylazine-induced loss of righting, which evaluates the ability to block central alpha(2)-adrenoceptors.

Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage

Telomeres are considered anti‐cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer‐prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53‐null K‐RasG12V‐induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long‐term whole‐body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.

A novel, facile methodology for the synthesis of N, N'-bis(tert-butoxycarbonyl)-protected guanidines using polymer-supported carbodiimide

A novel methodology for the synthesis of guanidines from amines has been developed using polymer assisted synthesis, potentially allowing the preparation of series of compounds in a high throughput manner. The methodology comprises the use of polymer-supported carbodiimide as the activating agent for N,N′-bis(tert-butoxycarbonyl) thiourea with polymer-supported trisamine as a scavenger, followed by deprotection with trifluoroacetic acid. For the first time, polymer-supported carbodiimide has been utilized as an activating agent to synthesize guanidines.

PI3Kα inhibition reduces obesity in mice

Partial inhibition of PI3K is one of the best-validated and evolutionary conserved manipulations to extend longevity. The best known health beneficial effects of reduced PI3K are related to metabolism and include increased energy expenditure, reduced nutrient storage, and protection from obesity. We have previously shown that a dual chemical inhibitor of the alpha and delta PI3K isoforms (CNIO-PI3Ki) reduces obesity in mice and monkeys, without evident toxic effects after long-term treatment. Here, we dissect the role of the alpha and delta PI3K isoforms by making use of selective inhibitors against PI3Kɑ (BYL-719 also known as alpelisib) or PI3Kδ (GS-9820 also known as acalisib). Treatment of mice with the above mentioned inhibitors indicated that BYL-719 increases energy expenditure in normal mice and efficiently reduces body weight in obese (ob/ob) mice, whereas these effects were not observed with GS-9820. Of note, the dose of BYL-719 required to reduce obesity was 10-times higher than the equivalent dose of CNIO-PI3Ki, which could suggest that simultaneous inhibition of PI3K alpha and delta is more beneficial than single inhibition of the alpha isoform. In summary, we conclude that inhibition of PI3Kɑ is sufficient to increase energy expenditure and reduce obesity, and suggest that concomitant PI3Kδ inhibition could play an auxiliary role.

Inhibition of TRF1 Telomere Protein Impairs Tumor Initiation and Progression in Glioblastoma Mouse Models and Patient-Derived Xenografts

Glioblastoma multiforme (GBM) is a deadly and common brain tumor. Poor prognosis is linked to high proliferation and cell heterogeneity, including glioma stem cells (GSCs). Telomere genes are frequently mutated. The telomere binding protein TRF1 is essential for telomere protection, and for adult and pluripotent stem cells. Here, we find TRF1 upregulation in mouse and human GBM. Brain-specific Trf1 genetic deletion in GBM mouse models inhibited GBM initiation and progression, increasing survival. Trf1 deletion increased telomeric DNA damage and reduced proliferation and stemness. TRF1 chemical inhibitors mimicked these effects in human GBM cells and also blocked tumor sphere formation and tumor growth in xenografts from patient-derived primary GSCs. Thus, targeting telomeres throughout TRF1 inhibition is an effective therapeutic strategy for GBM.

Abstract B228: Discovery of 6-Methyl-3-aryl-7,8-dihydro-6H-9-oxa-1,2,3a,4,6-pentaaza-cyclopenta[a]naphthalen-5-ylamine compounds as Pim kinase inhibitors with potent activity in tumoral cell lines and special binding mode.

Proviral integration site for Moloney murine leukemia virus (PIM) kinases are potent oncogens. Pim-1, 2 and 3 are highly conserved kinases that have unique structural properties, and are characterized by a constitutive serine/threonine activity that does not depend on post-translational modifications for activation. Pim kinases activity supports in vitro and in vivo tumor cell growth and survival through modification of an increasing number of common as well as isoform-specific substrates including c-myc and Histone H3, which drive transcription, eukaryotic elongation factor 4E-BP-1 that regulates transcription and Bad that activates cell survival. Furthermore, cell cycle protein activation by Pim kinases is involved with proliferation, and through regulation of AMPK activity Pim kinases mediated control of energy metabolism. Pim kinases are overexpressed in a range of haematopoietic malignancies and solid cancers, and its overexpression is associated with drug resistance. Inhibition of PIM kinase activity may be an attractive therapeutic strategy with possible favourable toxicity profiles due to minimal phenotype of mice mutant for all Pim family members. Knowledge of the three-dimensional structure of Pim1 is especially important for understanding the design of potent and selective Pim1 inhibitors as novel therapeutic agents. Using a rational strategy we have generated a novel chemical series, by introduction of a morpholine cycle in the C-7, C-8 position of the triazolopyridazine ring. Morpholine ring can be accommodated in the catalytic site due to a change in the conformation of the P-loop on the crystal structure to afford very potent and selective tricyclic compounds against PIM kinases. Depending on the amine C-6 substitution fragment we have observed different isoforms profiles. Here, we describe the exploration and biological characterization of C-6 tricyclic series, reporting its SAR/SPR (ADME). We identified lead compounds with potency in the low nanomolar range vs. PIM1, 2 and 3 and high selectivity versus a panel of 24 protein kinases. The compounds display cellular activity by blocking PIM signaling, S112 P-Bad in H1299 Pim1 cells, in the low nanomolar range. The combination of the PI3K inhibitor GDC-0941 with Pim inhibitors was strongly synergistic in vitro in non solid and solid tumoral cell lines. Finally, we assessed the effect of Pim kinase inhibition on Pim signaling in vivo in a mouse subcutaeneous tumor xenograft model employing a human mantle cell lymphoma cell line, Jeko-1. Results demonstrated that ETP-652 inhibited phosphorylation of S112 P-Bad in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B228.