Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

The importance and structural diversity of chiral amines is well‐demonstrated by the myriad nonenzymatic methods for their chemical production. In nature, the production of amines is performed by transamination rather than by reduction of an imine precursor derived from the corresponding ketone. Imine reductases, however, show great potential in the reduction of cyclic imines that are stable towards hydrolysis in aqueous reaction media. Here, we report the catalytic activity of two S‐selective imine reductases towards 3,4‐dihydroisoquinolines and 3,4‐dihydro‐β‐carbolines and their activity in the direct reductive amination of ketone substrates. The crystal structures of the enzyme from Streptomyces sp. GF3546 in complex with the cofactor NADPH and from Streptomyces aurantiacus in native form have been solved and refined to a resolution of 1.9 Å.

Bromodomains are acetyllysine epigenetic mark reader proteins. Small molecules inhibiting them have potential as anti‐inflammatory, antiviral, and anticancer agents. A 4‐acyl pyrrole derivative (see yellow structure in recognition pocket) has been identified that potently inhibits specific bromodomains and exhibits antiproliferative activity against leukemia cell lines.

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Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the ɛ-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making Sirt2 a promising target for pharmaceutical intervention. Here, based on a crystal structure of Sirt2 in complex with an optimized sirtuin rearranging ligand (SirReal) that shows improved potency, water solubility, and cellular efficacy, we present the development of the first Sirt2-selective affinity probe. A slow dissociation of the probe/enzyme complex offers new applications for SirReals, such as biophysical characterization, fragment-based screening, and affinity pull-down assays. This possibility makes the SirReal probe an important tool for studying sirtuin biology.

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Summary. The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer effiux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between I00 and 300 mM (25 ~ The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent haft-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30 mM and 400 mM respectively. Within the pH range of 6.3-8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaC1, NaNO3, Na-acetate, Na-lactate, Na-succinate or Na2HPO4). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37 ~ The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log Jso4 vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from -0.55 to -0.86, the slopes of the acidic branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyahate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a "two-site transport mechanism" consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.