Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors

Pharmacological activation of SIRT6 triggers lethal autophagy in human cancer cells

Sirtuin 6 (SIRT6) is a member of the NAD+-dependent class III deacetylase sirtuin family, which plays a key role in cancer by controlling transcription, genome stability, telomere integrity, DNA repair, and autophagy. Here we analyzed the molecular and biological effects of UBCS039, the first synthetic SIRT6 activator. Our data demonstrated that UBCS039 induced a time-dependent activation of autophagy in several human tumor cell lines, as evaluated by increased content of the lipidated form of LC3B by western blot and of autophagosomal puncta by microscopy analysis of GFP-LC3. UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity since the catalytic mutant H133Y failed to activate autophagy. At the molecular level, SIRT6-mediated autophagy was triggered by an increase of ROS levels, which, in turn, resulted in the activation of the AMPK-ULK1-mTOR signaling pathway. Interestingly, antioxidants were able to completely counteract UBCS039-induced autophagy, suggesting that ROS burst had a key role in upstream events leading to autophagy commitment. Finally, sustained activation of SIRT6 resulted in autophagy-related cell death, a process that was markedly attenuated using either a pan caspases inhibitor (zVAD-fmk) or an autophagy inhibitor (CQ). Overall, our results identified UBCS039 as an efficient SIRT6 activator, thereby providing a proof of principle that modulation of the enzyme can influence therapeutic strategy by enhancing autophagy-dependent cell death

The quinazoline ring as privileged scaffold in epigenetic medicinal chemistry

Our investigation of quinazolines as H3K9 methyltransferase/demethylase or DNMT inhibitors led us to identify MC3774, a Lys-mimicking derivative displaying dual G9a methyltransferase/LSD1 inhibition. In particular, MC3774 showed IC50 values of 1205 nM and 440 nM on G9a and LSD1 respectively. In MV4-11 leukemia cells, MC3774 displayed antiproliferative activity with IC50=894 nM. The most interesting aspect of this investigation is about the inhibition mechanism. We designed MC3774 supposing that the insertion of the propaneamine moiety could mimic the H3K4me2 moiety of the substrate bound within the active site. Surprisingly, the compound does not enter into the accessory pocket but binds the enzyme disposed with five copies in a stacked way obstructing the active site. The orientation of the molecules can be “face to face” or “head to tail”, and in both modes they interact with a cluster of negatively charged aminoacidic residues4. This kind of non-covalent and reversible inhibition is typical for the interaction between quinazoline scaffold and the catalytic cleft of LSD1, in fact MC3774 does not inhibit G9a using the same stacking mode. From the compound MC3774, some derivatives have been obtained as the result of the molecular simplification of the lead, which have led to the determination of the two methoxy at C6 and C7 positions, the dimethylaminopropylamine at C2 and the N-benzylpiperidine at C4, as essential for the effective inhibition of LSD1. Considering these observation, we worked on the synthesis of several analogs of the lead compound MC3774, modifying the quinazoline scaffold in position 2 with alkylamino functions of different length variously substituted at omega position and replacing the NH in position 4 with an oxygen atom or with N-CH3, or modifying the N-benzyl moiety with other aryl-alkyl functions, with the aim to increase the selectivity towards LSD1 or the dual inhibitory properties and to improve the potency in AML cells. These structural manipulations led to satisfying results: in fact two compounds among those synthesized, MC4016 and MC4057, show an increased inhibitory potency on LSD1 with Kd values in the nanomolar range. In addition, for the compound MC4013 we observed the improvement of the dual inhibitory properties demonstrated by the fact that the inhibitory potency grew on both the enzymes. Quinazolines also found a collocation among the wide array of DNMT inhibitors since when evidence of a low inhibitory activity of UNC0638 over DNMT1 was disclosed. In view of this finding, our research group spent many efforts in performing chemical manipulations of the quinazoline based histone methyltransferases and demethylases inhibitors, in order to achieve new knowledge about the potential of this scaffold in the context of DNMT inhibition. Firstly, we speculated that the removal of the methoxy groups from the positions 6 and 7 of the quinazoline core of BIX01294, UNC0638 and their analogs, should be useful to decrease the interactions that allow the targeting of enzymes that act on histone lysine residues. Indeed, the 6-methoxy group is mimic for H3A7 and the substituent at the C-7 reproduces the bound conformation of H3K9. Next, we investigated the substitution at the position 2 that led to the identification of MC2705 a 6,7-desmethoxyquinazoline equipped with the 4-amino-benzylpiperidine moiety and the 2-phenylpiperazine group at the C-2 position, as a novel and selective DNMT3A inhibitor. Another approach, was employed by Arimondo’s group. They supposed that the design of compounds able to resemble the transition state of the DNMT catalytic site, could be a good strategy to obtain an effective inhibition. Among the resulting series of derivatives, two compounds arranged with bulky substituents at the C-4 and equipped with cytosine mimic group the quinoline nucleus, linked to the C-7 through the 2-aminoethyl-piperidinemethanol moiety which showed high inhibitory potency against DNMT3A as attested by the IC50 values of 1.1 and 0.3 µM respectively. The present project arises from our fruitful collaboration with Arimondo’s group. The rational consists in the combination of the results obtained in the previous investigations, by designing new chimeric derivatives, endowed with the structural requirements that demonstrated to be essential for the inhibitory potency of the quinazoline based DNMT inhibitors, in order to get compounds suitable for the evaluation in vivo. The synthesis and biological evaluation of the designed series of properly substituted quinazoline based derivatives, led us to identify novel bulky compounds, endowed with excellent inhibitory potency against DNMT3A and, in a low extent, also against DNMT1. From a structural point of view, they are characterized by the quinazoline scaffold decorated with the 2-(N-phenylpiperazine), coming from the compound MC2705, the variously arranged bulky hydrophobic moiety at the position 4, connected to the central core by the crucial secondary amino group, and differently sized substituents at the position 7 ranging from the simple hydroxyl group to the cytidine-mimicking quinoline nucleus with its relative linker. The methoxy group at the position 6, whose presence was initially correlated with the enzymes involved in the histone methylation, fits well into the active site of DNMT3A, but it is not strictly essential, since its elimination does not affect the inhibitory potency. They were screened in KG-1 leukemic cells to evaluate their antiproliferative properties. The results indicated that some compounds among those synthesized, are able to inhibit the cells proliferation in a concentration dependent manner, with EC50 values, determined by measuring the level of ATP released by the viable cells, in the low micromolar range

Self-efficacy, postoperative care satisfaction, body image and sexual functioning in ARM patients

INTRODUCTION: Several studies indicate that self-efficacy may have profound positive effects on health promotion, clinical practice and other outcomes, like patients' satisfaction and quality of life. However no studies on self-efficacy have been conducted in anorectal malformation (ARM) patients. Aim of the present study is to analyze the relationships between self-efficacy and satisfaction with postoperative care in ARM patients or in their parents and between self-efficacy, body image and sexual functioning in adult ARM patients. MATERIAL AND METHODS: A total of 237 patients from 2 to 49 years old (mean age = 13.35 years old) participated in the study. Questionnaires were sent to members of the Italian Association for Anorectal Malformation (AIMAR) by ordinary mail. All subjects were asked to fill in the AIMAR questionnaire in which information about the perceived severity of malformation and postoperative care satisfaction were collected. For patients under 16 years old, parents were asked to fill in a questionnaire and a parent self-efficacy scale concerning the perceived ability to overcome possible difficulties related to ARM. Participants above 16 years of age were asked to fill in the questionnaire, a self-efficacy scale and some body image and sexual functioning items. RESULTS: The main findings indicate that those patients who feel more efficacious are those who feel less embarrassed and shame about their body, feel less frequently unsatisfied of their body and their physical condition, and have the perception to be less limited in their sexual activities. Considering patients under 16 years old, results indicate that more parents feel efficacious, the more they are satisfied of the postoperative care, especially of some health care providers. CONCLUSIONS: In ARM patients self-efficacy and parents self efficacy are correlated to important outcomes, respectively body image and sexual functioning in adults and postoperative care satisfaction in parents of those under 16 years old. Future longitudinal studies are needed to evaluate causal relations between the concepts considered

α,γ-Diketocarboxylic acids and their esters act as carbonic anhydrase IX and XII selective inhibitors

Among human carbonic anhydrase (CA) inhibitors, the α,γ-diketocarboxylic acids and esters are still poorly investigated. Here, we report the first compounds of this class (1-6) acting as potent inhibitors at low nanomolar level against the cancer-related human CA IX and XII, and 2-3 magnitude orders selective toward the cytosolic isoforms hCA I and II. At enzymatic level, the α,γ-diketoacids 1-3 were more effective inhibitors compared to the corresponding ethyl esters 4-6. The phenyl- and α-naphthyl-containing compounds (1, 3, 4, and 6) behaved as dual hCA IX/XII inhibitors, while the β-naphthyl analogues (2 and 5) exhibited hCA IX-selective inhibition. In MG63 and HOS osteosarcoma (OS) cell lines, the ethyl esters 5 and 6 displayed dose-dependent reduction of viability and proliferation after 72 h treatment, with 6 being more potent than 5 likely for its dual hCA IX/XII inhibition