Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia.

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

In vitro and in vivo induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor.

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2'-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.

The comparison of frequency of the upper limb musculoskeletal disorders among patients with diabetes type II with normal cases.

BACKGROUND AND AIM: Musculoskeletal disease, that is recognized in diabetes and diabetes mellitus (DM) has shown a higher prevalence of chronic musculoskeletal complications. This study aimed at assessing the frequency of upper limb musculoskeletal disorders among patients with diabetes type II with normal cases in Mashhad, Iran. METHODS: A cross-section of 100 patients with upper limb musculoskeletal disorders were enrolled in this study. The patients were examined by a unique physician considering carpal tunnel syndrome disorder, trigger finger, adhesive capsulitis, and Dupuytren's contracture at Ghaem hospital, Mashhad, Iran in 2015. All collected data were recorded by using SPSS version 21 and were analyzed through independent-samples t-test for comparing changes, and Chi-square. RESULTS: In this study, the mean age was 51.7±8.7 years old. Gender frequency was 114 (57%) male, and 86 (43%) female. There was no significant difference between groups in cases of gender frequency and mean of age (p>0.05). In evaluation of association between the two groups, there was significant difference for adhesive capsulitis, (p=0.04). Chi-square test showed significant association for age and adhesive capsulitis between the two groups, (p=0.040); but no other diabetes-related disorders, (p<0.05). CONCLUSION: The results of this study showed that in patients with diabetes mellitus and musculoskeletal complications such as upper limb musculoskeletal abnormalities, it will lead to an increase in skeletal muscle effects in DM patients. It is recommended that musculoskeletal examination is done periodically in DM patients for identification of these disorders and necessary actions are carried out for prevention of the disorders as soon as possible.

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a 5'-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II (TOP2). TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for high-throughput screen (HTS)-screening. We have gone on to determine crystal structures of these compounds bound to a 'humanized' form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2.

Can the inpatient hospital setting be a golden opportunity to improve colon cancer screening rates in the United States?

BACKGROUND: Colorectal cancer is the third most common cancer in the United States. Despite efforts to increase colorectal cancer screening, the rate of compliance with the recommended screening remains relatively suboptimal according the American Cancer Society (53%). PURPOSE: To assess whether the time of hospitalization is a suitable opportunity for patients to receive counseling and for recruiting patients to undergo screening colonoscopy for colon cancer. METHOD: In 2009, we conducted a cross-sectional survey of hospitalized adults age 50 to 80 years in order to assess their responses on a modified version of the Health Information National Trends Survey. We conducted χ(2) analyses on these data to examine the differences in patients' knowledge of colorectal cancer screening and prior adherence to screening guidelines and to assess whether they would be willing to undergo a screening in the near future if prompted by their physicians. RESULTS: We enrolled a total of 332 participants to complete the study questionnaire. About 94% of the subjects had heard about colon cancer, and 83.4% had heard of any screening tests to detect colorectal cancer. About 66% of subjects reported the colonoscopy to be the most effective screening test for colon cancer. Approximately 55% of the total sample group adhered to recommended screening guidelines for colon cancer using the colonoscopy. CONCLUSIONS: The time of hospitalization is a potential "golden opportunity" to counsel patients and promote colon cancer screening.

Toxoflavins and deazaflavins as the first reported selective small molecule inhibitors of tyrosyl-DNA phosphodiesterase II.

The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.

Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.

Topoisomerases regulate DNA topology by the transient cleavage and religation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase that repairs this topoisomerase-mediated DNA damage, thereby playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in increased susceptibility and sensitivity to Topo II-induced DNA double-strand breaks, thereby revealing Tdp2 as a potentially attractive anticancer target. No drug-like inhibitors of Tdp2 have been identified to date, and assays suitable for high-throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogeneous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. In addition, a new and specific Tdp2 antibody is described. Together, these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.

Effectiveness and safety of raloxifene in post-menopausal females.

OBJECTIVE: To determine effects of raloxifene on lipid profile, breast tissue and endometrial thickness in post-menopausal women. DESIGN: A longitudinal cohort study. PLACE AND DURATION OF STUDY: The study was conducted at Department of Gynaecology and Obstetrics, PNS Shifa Hospital, Karachi over a period of one year. PATIENTS AND METHODS: Fifty cases of postmenopausal women, aged 45-60 years, who had last menstrual period 2-8 years back, visiting Gynea OPD at PNS Shifa Hospital, Karachi, were included in the study. The subject received raloxifene (Evista) 60 mg once daily. The tests done at the start of study as baseline were serum lipid profile (HDL, LDL, total triglyceride and cholesterol levels), TVS (trans vaginal sonography) for endometrial thickness and mammography. The above tests were again done at the end of study after 12 months. The adverse effects were recorded. RESULTS: Fifty women were enrolled for study out of which 48 completed the study that is 96%. Mean age was 54 years, SD = 3.95. The mean value of LDL, total triglyceride and cholesterol decreased by 15.2%, 1.2% and 10.2 % respectively. The change was statistically significant. (p < 0.001) HDL change was 0.665% and was not statistically significant. TVS, for endometrial thickness, and mammography for breast density showed no change. Adverse effects reported were hot flushes, leg cramps and vaginal symptoms in few cases. CONCLUSION: Raloxifene therapy significantly decreases cardiovascular risk markers LDL, total triglyceride and cholesterol. No abnormal change in breast density and in endometrial thickness indicate good safety profile of this agent.

Synthesis of enantiopure dihydropyranones: aldol-based ring expansion of dihydrofurans.

[reaction: see text] The stereoselective Birch reduction of 3-methyl-2-furoic acids using a readily available chiral auxilairy is described; by coupling this process to an oxidative cleavage/aldol ring closure sequence we were able to produce highly functionalized and enantiopure dihydropyranones in high yield. This sequence has ample flexibility built into it, either by the use of different electrophiles during reductive alkylation or by subsequent derivatization of the dihydropyranone after ring expansion.