Rational Design of Azastatin as a Potential ADC Payload with Reduced Bystander Killing.

Auristatins are a class of ultrapotent microtubule inhibitors, whose growing clinical popularity in oncology is based upon their use as payloads in antibody-drug conjugates (ADCs). The most widely utilized auristatin, MMAE, has however been shown to cause apoptosis in non-pathological cells proximal to the tumour ("bystander killing"). Herein, we introduce azastatins, a new class of auristatin derivatives encompassing a side chain amine for antibody conjugation. The synthesis of Cbz-azastatin methyl ester, which included the C2-elongation and diastereoselective reduction of two proteinogenic amino acids as key transformations, was accomplished in 22 steps and 0.76 % overall yield. While Cbz-protected azastatin methyl ester (0.13-3.0 nM) inhibited proliferation more potently than MMAE (0.47-6.5 nM), removal of the Cbz-group yielded dramatically increased IC50 -values (9.8-170 nM). We attribute the reduced apparent cytotoxicity of the deprotected azastatin methyl esters to a lack of membrane permeability. These results clearly establish the azastatins as a novel class of cytotoxic payloads ideally suited for use in next-generation ADC development.

Human MRP2 exports MC-LR but not the glutathione conjugate.

Water contamination by cyanobacterial blooms is a worldwide health hazard to humans as well as livestock. Exposure to Microcystins (MCs), toxins produced by various cyanobacterial or blue green algae found in poorly treated drinking water or contaminated seafood such as fish or prawns are associated with hepatotoxicity, nephropathy and neurotoxicity and in extreme cases, death in humans. MC congeners, currently >240 known, differ dramatically in their uptake kinetics, i.e. their uptake via OATP1B1 and OATP1B3, in OATP overexpressing human HEK293 cells and primary human hepatocytes. It is thus likely that MC congeners will also differ with respect to the cellular efflux of the parent and conjugated congeners, e.g. via MRPs, MDRs, BCRP or BSEP. Consequently, the role and kinetics of different human efflux transporters - MRP, MDR, BCRP and BSEP in MC efflux was studied using insect membrane vesicles overexpressing the human transporters of interest. Of the efflux transporters investigated, MRP2 displayed MC transport. Michaelis-Menten kinetics displayed mild co-operativity and thus allosteric behavior of MRP2. MC transport by MRP2 was MC congener-specific, whereby MC-LF was transported more rapidly than MC-LR and -RR. Other human transporters (BCRP, BSEP, MRP1,3,5, MDR1) tested in this study did not exhibit interaction with MC. Although MRP2 showed specific MC transport, the MC-LR-GSH conjugate, was not transported suggesting the involvement of other transporters than MRP2 for the conjugate efflux.

Triple Orthogonal Labeling of Glycans by Applying Photoclick Chemistry.

Bioorthogonal labeling of multiple biomolecules is of current interest in chemical biology. Metabolic glycoengineering (MGE) has been shown to be an appropriate approach to visualizing carbohydrates. Here, we report that the nitrile imine-alkene cycloaddition (photoclick reaction) is a suitable ligation reaction in MGE. Using a mannosamine derivative with an acrylamide reporter group that is efficiently metabolized by cells and that quickly reacts in the photoclick reaction, we labeled sialic acids on the surface of living cells. Screening of several alkenes showed that a previously reported carbamate-linked methylcyclopropene reporter that is well suited for the inverse-electron-demand Diels-Alder (DAinv ) reaction has a surprisingly low reactivity in the photoclick reaction. Thus, for the first time, we were able to triply label glycans by a combination of DAinv , photoclick, and copper-free click chemistry.