Discovery of an MLLT1/3 YEATS Domain Chemical Probe.

YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine-binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small-molecule chemical probe, SGC-iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC-iMLLT is a potent and selective inhibitor of MLLT1/3-histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small-molecule X-ray co-crystal structures with the MLLT1 YD are also reported. This first-in-class probe molecule can be used to understand MLLT1/3-associated biology and the therapeutic potential of small-molecule YD inhibitors.

A role for the metalloprotease invadolysin in insulin signaling and adipogenesis.

Invadolysin is a novel metalloprotease conserved amongst metazoans that is essential for life in Drosophila. We previously showed that invadolysin was essential for the cell cycle and cell migration, linking to metabolism through a role in lipid storage and interaction with mitochondrial proteins. In this study we demonstrate that invadolysin mutants exhibit increased autophagy and decreased glycogen storage - suggestive of a role for invadolysin in insulin signaling in Drosophila. Consistent with this, effectors of insulin signaling were decreased in invadolysin mutants. In addition, we discovered that invadolysin was deposited on newly synthesized lipid droplets in a PKC-dependent manner. We examined two in vitro models of adipogenesis for the expression and localization of invadolysin. The level of invadolysin increased during both murine 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS), adipogenesis. Invadolysin displayed a dynamic localization to lipid droplets over the course of adipogenesis, which may be due to the differential expression of distinct invadolysin variants. Pharmacological inhibition of adipogenesis abrogated the increase in invadolysin. In summary, our results on in vivo and in vitro systems highlight an important role for invadolysin in insulin signaling and adipogenesis.

Invadolysin acts genetically via the SAGA complex to modulate chromosome structure.

Identification of components essential to chromosome structure and behaviour remains a vibrant area of study. We have previously shown that invadolysin is essential in Drosophila, with roles in cell division and cell migration. Mitotic chromosomes are hypercondensed in length, but display an aberrant fuzzy appearance. We additionally demonstrated that in human cells, invadolysin is localized on the surface of lipid droplets, organelles that store not only triglycerides and sterols but also free histones H2A, H2Av and H2B. Is there a link between the storage of histones in lipid droplets and the aberrantly structured chromosomes of invadolysin mutants? We have identified a genetic interaction between invadolysin and nonstop, the de-ubiquitinating protease component of the SAGA (Spt-Ada-Gcn5-acetyltransferase) chromatin-remodelling complex. invadolysin and nonstop mutants exhibit phenotypic similarities in terms of chromosome structure in both diploid and polyploid cells. Furthermore, IX-14(1)/not(1) transheterozygous animals accumulate mono-ubiquitinated histone H2B (ubH2B) and histone H3 tri-methylated at lysine 4 (H3K4me3). Whole mount immunostaining of IX-14(1)/not(1) transheterozygous salivary glands revealed that ubH2B accumulates surprisingly in the cytoplasm, rather than the nucleus. Over-expression of the Bre1 ubiquitin ligase phenocopies the effects of mutating either the invadolysin or nonstop genes. Intriguingly, nonstop and mutants of other SAGA subunits (gcn5, ada2b and sgf11) all suppress an invadolysin-induced rough eye phenotype. We conclude that the abnormal chromosome phenotype of invadolysin mutants is likely the result of disrupting the histone modification cycle, as accumulation of ubH2B and H3K4me3 is observed. We further suggest that the mislocalization of ubH2B to the cytoplasm has additional consequences on downstream components essential for chromosome behaviour. We therefore propose that invadolysin plays a crucial role in chromosome organization via its interaction with the SAGA complex.

Creation of a Novel Class of Potent and Selective MutT Homologue 1 (MTH1) Inhibitors Using Fragment-Based Screening and Structure-Based Drug Design.

Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo. (1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment-based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.

Influence of temperature, needle gauge and injection rate on the size distribution, concentration and acoustic responses of ultrasound contrast agents at high frequency.

This paper investigated the influence of needle gauge (19G and 27G), injection rate (0.85ml·min(-1), 3ml·min(-1)) and temperature (room temperature (RT) and body temperature (BT)) on the mean diameter, concentration, acoustic attenuation, contrast to tissue ratio (CTR) and normalised subharmonic intensity (NSI) of three ultrasound contrast agents (UCAs): Definity, SonoVue and MicroMarker (untargeted). A broadband substitution technique was used to acquire the acoustic properties over the frequency range 17-31MHz with a preclinical ultrasound scanner Vevo770 (Visualsonics, Canada). Significant differences (P<0.001-P<0.05) between typical in vitro setting (19G needle, 3ml·min(-1) at RT) and typical in vivo setting (27G needle, 0.85ml·min(-1) at BT) were found for SonoVue and MicroMarker. Moreover we found that the mean volume-based diameter and concentration of both SonoVue and Definity reduced significantly when changing from typical in vitro to in vivo experimental set-ups, while those for MicroMarker did not significantly change. From our limited measurements of Definity, we found no significant change in attenuation, CTR and NSI with needle gauge. For SonoVue, all the measured acoustic properties (attenuation, CTR and NSI) reduced significantly when changing from typical in vitro to in vivo experimental conditions, while for MicroMarker, only the NSI reduced, with attenuation and CTR increasing significantly. These differences suggest that changes in physical compression and temperature are likely to alter the shell structure of the UCAs resulting in measureable and significant changes in the physical and high frequency acoustical properties of the contrast agents under typical in vitro and preclinical in vivo experimental conditions.