Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells.

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Targeting of the type II inositol polyphosphate 5-phosphatase INPP5B to the early secretory pathway.

The inositol polyphosphate 5-phosphatase INPP5B is closely related to the Lowe syndrome protein OCRL1, sharing a similar substrate specificity, domain organisation and an ability to compensate for loss of OCRL1 in knockout mice. The cellular localisation and functions of INPP5B have remained poorly defined until recently, when a role within the endocytic pathway was suggested. Here, we report that INPP5B is also localised to the early secretory pathway including the Golgi apparatus and ER-to-Golgi intermediate compartment (ERGIC). Consistent with this localisation, INPP5B binds to specific RAB proteins within the secretory pathway, and mutational analysis indicates that RAB binding is required for efficient Golgi targeting of INPP5B. Unlike OCRL1, INPP5B interacts with neither clathrin nor alpha-adaptin and is largely absent from clathrin-coated intermediates. Expression of INPP5B but not OCRL1 alters the distribution of the cycling protein ERGIC53 when cells are incubated at low temperature (15 degrees C) or in the presence of brefeldin A, causing ERGIC53 to accumulate in the ERGIC, with a concomitant loss from the ER. Our data suggest a role for INPP5B in retrograde ERGIC-to-ER transport and imply that it has functions distinct from those of OCRL1 within both the secretory and endocytic pathways.

Membrane targeting and activation of the Lowe syndrome protein OCRL1 by rab GTPases.

The X-linked disorder oculocerebrorenal syndrome of Lowe is caused by mutation of the OCRL1 protein, an inositol polyphosphate 5-phosphatase. OCRL1 is localised to the Golgi apparatus and early endosomes, and can translocate to lamellipodia upon growth factor stimulation. We show here that OCRL1 interacts with several members of the rab family of small GTPases. Strongest interaction is seen with Golgi-associated rab1 and rab6 and endosomal rab5. Point mutants defective in rab binding fail to target to the Golgi apparatus and endosomes, strongly suggesting rab interaction is required for targeting of OCRL1 to these compartments. Membrane recruitment via rab binding is required for changes in Golgi and endosomal dynamics induced by overexpression of catalytically inactive OCRL1. In vitro experiments demonstrate that rab5 and rab6 directly stimulate the 5-phosphatase activity of OCRL1. We conclude that rabs play a dual role in regulation of OCRL1, firstly targeting it to the Golgi apparatus and endosomes, and secondly, directly stimulating the 5-phosphatase activity of OCRL1 after membrane recruitment.