ML216

IC50: 3.0 and 0.97 μM for full length BLM and BLM636–1298, respectively

ML216 is a potent inhibitor of the DNA unwinding activity of BLM helicase.

BLM helicase is reported to be a DNA unwinding enzyme critical in DNA repair through the homologous recombination pathway. BLM gene mutations lead to diminished BLM helicase activity and can cause Bloom’s Syndrome. Similar to other DNA repair enzymes, BLM helicase inhibition shows sensitization of tumor cells to conventional cancer therapies, such as camptothecin.

In vitro: ML216 showed submicromolar potency and selectivity over related helicases including RECQ1, RECQ5, and E. coli UvrD helicases. ML216 also inhibited cell proliferation of BLM-proficient fibroblast cells while had minimal effects on BLM deficient fibroblast cells, indicating on-target activity in a cellular context. Additionally, ML216 increased the frequency of sister chromatid exchanges, which was a diagnostic cellular phenotype consistent with the absence of a functional BLM protein [1].

In vivo: ML216 was a suitable starting point for further mouse tumor xenograft models and for the further development of potential cancer therapeutics [1].

Clinical trial: N/A

Reference:
[1] Rosenthal AS,Dexheimer TS,Nguyen G,Gileadi O,Vindigni A,Simeonov A,Jadhav A,Hickson I,Maloney DJ.  Discovery of ML216, a Small Molecule Inhibitor of Bloom (BLM) Helicase. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-2011 Apr 15

Genetic mapping of species differences via in vitro crosses in mouse embryonic stem cells.[Pubmed:29563231]

Proc Natl Acad Sci U S A. 2018 Apr 3;115(14):3680-3685.

Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic mapping of trait variation across species. Here, we describe a simple, robust, and transgene-free technique to generate "in vitro crosses" in hybrid mouse embryonic stem (ES) cells by inducing random mitotic cross-overs with the drug ML216, which inhibits the DNA helicase Bloom syndrome (BLM). Starting with an interspecific F1 hybrid ES cell line between the Mus musculus laboratory mouse and Mus spretus ( approximately 1.5 million years of divergence), we mapped the genetic basis of drug resistance to the antimetabolite tioguanine to a single region containing hypoxanthine-guanine phosphoribosyltransferase (Hprt) in as few as 21 d through "flow mapping" by coupling in vitro crosses with fluorescence-activated cell sorting (FACS). We also show how our platform can enable direct study of developmental variation by rederiving embryos with contribution from the recombinant ES cell lines. We demonstrate how in vitro crosses can overcome major bottlenecks in mouse complex trait genetics and address fundamental questions in evolutionary biology that are otherwise intractable through traditional breeding due to high cost, small litter sizes, and/or hybrid sterility. In doing so, we describe an experimental platform toward studying evolutionary systems biology in mouse and potentially in human and other mammals, including cross-species hybrids.

Pacemaker role of pericytes in generating synchronized spontaneous Ca2+ transients in the myenteric microvasculature of the guinea-pig gastric antrum.[Pubmed:26153078]

Cell Calcium. 2015 Nov;58(5):442-56.

Properties of spontaneous Ca(2+) transients in the myenteric microvasculature of the guinea-pig stomach were investigated. Specifically, we explored the spatio-temporal origin of Ca(2+) transients and the role of voltage-dependent Ca(2+) channels (VDCCs) in their intercellular synchrony using fluorescence Ca(2+) imaging and immunohistochemistry. The microvasculature generated spontaneous Ca(2+) transients that were independent of both Ca(2+) transients in interstitial cells of Cajal (ICC) and neural activity. Spontaneous Ca(2+) transients were highly synchronous along the length of microvasculature, and appeared to be initiated in pericytes and spread to arteriolar smooth muscle cells (SMCs). In most cases, the generation or synchrony of Ca(2+) transients was not affected by blockers of L-type VDCCs. In nifedipine-treated preparations, synchronous spontaneous Ca(2+) transients were readily blocked by Ni(2+), mibefradil or ML216, blockers for T-type VDCCs. These blockers also suppressed the known T-type VDCC dependent component of ICC Ca(2+) transients or slow waves. Spontaneous Ca(2+) transients were also suppressed by caffeine, tetracaine or cyclopiazonic acid (CPA). After the blockade of both L- and T-type VDCCs, asynchronous Ca(2+) transients were generated in pericytes on precapillary arterioles and/or capillaries but not in arteriolar SMCs, and were abolished by CPA or nominally Ca(2+) free solution. Together these data indicate that pericytes in the myenteric microvasculature may act as the origin of synchronous spontaneous Ca(2+) transients. Pericyte Ca(2+) transients arise from Ca(2+) release from the sarco-endoplasmic reticulum and the opening of T-type Ca(2+) VDCCs is required for their synchrony and propagation to arteriolar SMCs.

Synthesis and SAR studies of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine derivatives as potent inhibitors of Bloom helicase.[Pubmed:24012121]

Bioorg Med Chem Lett. 2013 Oct 15;23(20):5660-6.

Human cells utilize a variety of complex DNA repair mechanisms in order to combat constant mutagenic and cytotoxic threats from both exogenous and endogenous sources. The RecQ family of DNA helicases, which includes Bloom helicase (BLM), plays an important function in DNA repair by unwinding complementary strands of duplex DNA as well as atypical DNA structures such as Holliday junctions. Mutations of the BLM gene can result in Bloom syndrome, an autosomal recessive disorder associated with cancer predisposition. BLM-deficient cells exhibit increased sensitivity to DNA damaging agents indicating that a selective BLM inhibitor could be useful in potentiating the anticancer activity of these agents. In this work, we describe the medicinal chemistry optimization of the hit molecule following a quantitative high-throughput screen of >355,000 compounds. These efforts lead to the identification of ML216 and related analogs, which possess potent BLM inhibition and exhibit selectivity over related helicases. Moreover, these compounds demonstrated cellular activity by inducing sister chromatid exchanges, a hallmark of Bloom syndrome.

A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells.[Pubmed:23352139]

Chem Biol. 2013 Jan 24;20(1):55-62.

The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM function in cells, we undertook a high throughput screen of a chemical compound library for small molecule inhibitors of BLM. We present ML216, a potent inhibitor of the DNA unwinding activity of BLM. ML216 shows cell-based activity and can induce sister chromatid exchanges, enhance the toxicity of aphidicolin, and exert antiproliferative activity in cells expressing BLM, but not those lacking BLM. These data indicate that ML216 shows strong selectivity for BLM in cultured cells. We discuss the potential utility of such a BLM-targeting compound as an anticancer agent.

ML216 (CID-49852229) is a potent, selective and cell permeable inhibitor of the DNA unwinding activity of BLM helicase with IC50s of 2.98 μM and 0.97 μM for BLMfull-length and BLM636-1298, respectively. ML216 inhibits ssDNA-dependent ATPase activity of BLM with a Ki of 1.76 µM. Antitumor avtivity.

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