Mapping the substrate specificity of the Plasmodium M1 and M17 aminopeptidases.

During malarial infection, Plasmodium parasites digest human hemoglobin to obtain free amino acids for protein production and maintenance of osmotic pressure. The Plasmodium M1 and M17 aminopeptidases are both postulated to have an essential role in the terminal stages of the hemoglobin digestion process and are validated drug targets for the design of new dual-target anti-malarial compounds. In this study, we profiled the substrate specificity fingerprints and kinetic behaviors of M1 and M17 aminopeptidases from Plasmodium falciparum and Plasmodium vivax, and the mouse model species, Plasmodium berghei. We found that although the Plasmodium M1 aminopeptidases share a largely similar, broad specificity at the P1 position, the P. falciparum M1 displays the greatest diversity in specificity and P. berghei M1 showing a preference for charged P1 residues. In contrast, the Plasmodium M17 aminopeptidases share a highly conserved preference for hydrophobic residues at the P1 position. The aminopeptidases also demonstrated intra-peptide sequence specificity, particularly the M1 aminopeptidases, which showed a definitive preference for peptides with fewer negatively charged intrapeptide residues. Overall, the P. vivax and P. berghei enzymes had a faster substrate turnover rate than the P. falciparum enzymes, which we postulate is due to subtle differences in structural dynamicity. Together, these results build a kinetic profile that allows us to better understand the catalytic nuances of the M1 and M17 aminopeptidases from different Plasmodium species.

Diagnostic and therapeutic potential of protease inhibition.

Proteases are enzymes that hydrolyze peptide bonds in proteins and peptides; thus, they control virtually all biological processes. Our understanding of protease function has advanced considerably from nonselective digestive enzymes to highly specialized molecular scissors that orchestrate complex signaling networks through a limited proteolysis. The catalytic activity of proteases is tightly regulated at several levels, ranging from gene expression through trafficking and maturation to posttranslational modifications. However, when this delicate balance is disturbed, many diseases develop, including cancer, inflammatory disorders, diabetes, and neurodegenerative diseases. This new understanding of the role of proteases in pathologic physiology indicates that these enzymes represent excellent molecular targets for the development of therapeutic inhibitors, as well as for the design of chemical probes to visualize their redundant activity. Recently, numerous platform technologies have been developed to identify and optimize protease substrates and inhibitors, which were further used as lead structures for the development of chemical probes and therapeutic drugs. Due to this considerable success, the clinical potential of proteases in therapeutics and diagnostics is rapidly growing and is still not completely explored. Therefore, small molecules that can selectively target aberrant protease activity are emerging in diseases cells. In this review, we describe modern trends in the design of protease drugs as well as small molecule activity-based probes to visualize selected proteases in clinical settings.

Current methods for the synthesis of homogeneous antibody-drug conjugates.

Development of efficient and safe cancer therapy is one of the major challenges of the modern medicine. Over the last few years antibody-drug conjugates (ADCs) have become a powerful tool in cancer treatment with two of them, Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine), having recently been approved by the Food and Drug Administration (FDA). Essentially, an ADC is a bioconjugate that comprises a monoclonal antibody that specifically binds tumor surface antigen and a highly potent drug, which is attached to the antibody via either cleavable or stable linker. This approach ensures specificity and efficacy in fighting cancer cells, while healthy tissues remain largely unaffected. Conventional ADCs, that employ cysteine or lysine residues as conjugation sites, are highly heterogeneous. This means that the species contain various populations of the ADCs with different drug-to-antibody ratios (DARs) and different drug load distributions. DAR and drug-load distribution are essential parameters of ADCs as they determine their stability and efficacy. Therefore, various drug-loaded forms of ADCs (usually from zero to eight conjugated molecules per antibody) may have distinct pharmacokinetics (PK) in vivo and may differ in clinical performance. Recently, a significant progress has been made in the field of site-specific conjugation which resulted in a number of strategies for synthesis of the homogeneous ADCs. This review describes newly-developed methods that ensure homogeneity of the ADCs including use of engineered reactive cysteine residues (THIOMAB), unnatural amino acids, aldehyde tags, enzymatic transglutaminase- and glycotransferase-based approaches and novel chemical methods. Furthermore, we briefly discuss the limitation of these methods emphasizing the need for further improvement in the ADC design and development.