CE methods for charge variant analysis of mAbs and complex format biotherapeutics.

Charge heterogeneity analysis of monoclonal antibodies (mAbs) and complex formats, such as bispecifics, is crucial for therapeutic applications. In this study, we developed two capillary electrophoresis (CE)-based methods, capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF), for analyzing a broad spectrum of mAbs and complex mAb formats. For CZE, we introduced a new buffer system and optimized the background electrolyte (BGE) with an alternative dynamic coating agent and a superior polymeric additive. The pH of the BGE was increased, leading to enhanced resolution of high pI and complex format mAbs. In iCIEF, we identified an ampholyte combination offering a highly linear pH gradient and covering a suitable pH range. We also investigated alternatives to denaturing stabilizers and found that non-detergent sulfobetaine 195 exhibited excellent properties for iCIEF applications. These optimized methods provide a framework for the charge heterogeneity analysis of therapeutic mAbs and complex formats.

Specific Inhibitors of Mitochondrial Deacylase Sirtuin 4 Endowed with Cellular Activity.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in aging-related diseases. Mammalian Sirtuin 4 (Sirt4) is located in mitochondria and a potential therapeutic target for cancer and metabolic diseases, but no potent and selective Sirt4 inhibitors have been reported. Here, we describe the identification of potent Sirt4-specific small-molecule inhibitors. Testing hits from a target-based virtual screen revealed 12 active compounds. A focused screen based on two top compounds, followed by structure-assisted design of derivatives, yielded four first-in-class potent Sirt4 inhibitors. Kinetic analyses indicate compound competition with the acyl peptide substrate, consistent with the docking models and implicating Sirt4's unique acyl binding site. The compounds indeed show preference for Sirt4 over other isoforms, with one of them (69) being highly isoform selective, and they are active in cells. Our results provide first lead compounds and mechanistic insights for optimization toward Sirt4-specific inhibitors useful as experimental tools and potential therapeutics.

From PARP1 to TNKS2 Inhibition: A Structure-Based Approach.

Tankyrases (TNKSs) have recently gained great consideration as potential targets in Wnt/ß-catenin pathway-dependent solid tumors. Previously, we reported the 2-mercaptoquinazolin-4-one MC2050 as a micromolar PARP1 inhibitor. Here we show how the resolution of the X-ray structure of PARP1 in complex with MC2050, combined with the computational investigation of the structural differences between TNKSs and PARP1/2 active sites, provided the rationale for a structure-based drug design campaign that with a limited synthetic effort led to the discovery of the bis-quinazolinone 5 as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated.

Potent and Selective Inhibitors of Human Sirtuin 5.

Sirtuins are protein deacylases that regulate metabolism and stress responses and are implicated in aging-related diseases. Modulators of the human sirtuins Sirt1-7 are sought as chemical tools and potential therapeutics, e.g., for cancer. Selective and potent inhibitors are available for Sirt2, but selective inhibitors for Sirt5 with Ki values in the low nanomolar range are lacking. We synthesized and screened 3-arylthiosuccinylated and 3-benzylthiosuccinylated peptide derivatives yielding Sirt5 inhibitors with low-nanomolar Ki values. A biotinylated derivative with this scaffold represents an affinity probe for human Sirt5 that is able to selectively extract this enzyme out of complex biological samples like cell lysates. Crystal structures of Sirt5/inhibitor complexes reveal that the compounds bind in an unexpected manner to the active site of Sirt5.

Crystal structures of the mitochondrial deacylase Sirtuin 4 reveal isoform-specific acyl recognition and regulation features.

Sirtuins are evolutionary conserved NAD+-dependent protein lysine deacylases. The seven human isoforms, Sirt1-7, regulate metabolism and stress responses and are considered therapeutic targets for aging-related diseases. Sirt4 locates to mitochondria and regulates fatty acid metabolism and apoptosis. In contrast to the mitochondrial deacetylase Sirt3 and desuccinylase Sirt5, no prominent deacylase activity and structural information are available for Sirt4. Here we describe acyl substrates and crystal structures for Sirt4. The enzyme shows isoform-specific acyl selectivity, with significant activity against hydroxymethylglutarylation. Crystal structures of Sirt4 from Xenopus tropicalis reveal a particular acyl binding site with an additional access channel, rationalizing its activities. The structures further identify a conserved, isoform-specific Sirt4 loop that folds into the active site to potentially regulate catalysis. Using these results, we further establish efficient Sirt4 activity assays, an unusual Sirt4 regulation by NADH, and Sirt4 effects of pharmacological modulators.

Mechanism-Based Inhibitors of the Human Sirtuin 5 Deacylase: Structure-Activity Relationship, Biostructural, and Kinetic Insight.

The sirtuin enzymes are important regulatory deacylases in a variety of biochemical contexts and may therefore be potential therapeutic targets through either activation or inhibition by small molecules. Here, we describe the discovery of the most potent inhibitor of sirtuin 5 (SIRT5) reported to date. We provide rationalization of the mode of binding by solving co-crystal structures of selected inhibitors in complex with both human and zebrafish SIRT5, which provide insight for future optimization of inhibitors with more "drug-like" properties. Importantly, enzyme kinetic evaluation revealed a slow, tight-binding mechanism of inhibition, which is unprecedented for SIRT5. This is important information when applying inhibitors to probe mechanisms in biology.

Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

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.

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5.