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Keywords: ethinylestradiol; lysyl hydroxylase 2; target identification; yeast three-hybrid screen EN ethinylestradiol lysyl hydroxylase 2 target identification yeast three-hybrid screen 421 431 11 03/11/22 20220301 NES 220301 Introduction Many fundamental processes in biology rely on small ligand-protein interactions which form the basis for pharmacological intervention of human diseases. After washing, bound proteins were eluted with SDS and heat, and analyzed by SDS-PAGE and Western Blotting using a PLOD2 antibody for staining, or a GAL4 antibody for the detection of the GAL4 AD fusion protein from the Y3H vector. Having confirmed that our Y3H setup can indeed detect small molecule-protein interactions, we next wanted to employ this system for the identification of protein binding partners of small molecules out of a pool of proteins. [Extracted from the article]

Extensive studies have reported that interaction of α-synuclein amyloid species with neurons is a crucial mechanistic characteristic of Parkinson's disease (PD) and small molecules can downregulate the neurotoxic effects induced by protein aggregation. However, the exact mechanism(s) of these neuroprotective effects by small molecules remain widely unknown. In the present study, α-synuclein samples in the amyloidogenic condition were aged for 120 h with or without different concentrations of mitoquinone (MitoQ) as a quinone derivative compound and the amyloid characteristics and the relevant neurotoxicity were evaluated by Thioflavin T (ThT)/Nile red fluorescence, Congo red absorption, circular dichroism (CD), transmission electron microscopy (TEM), cell viability, lactate dehydrogenase (LDH), reactive oxygen species (ROS), reactive nitrogen species (RNS), malondialdehyde (MDA), superoxide dismutase (SOD), and caspase-9/-3 activity assays. Results clearly showed the capacity of MitoQ on the inhibition of the formation of α-synuclein fibrillation products through modulation of the aggregation pathway by an effect on the kinetic parameters. Also, it was shown that α-synuclein samples aged for 120 h with MitoQ trigger less neurotoxic effects against SH-SY5Y cells than α-synuclein amyloid alone. Indeed, co-incubation of α-synuclein with MitoQ reduced the membrane leakage, oxidative and nitro-oxidative stress, modifications of macromolecules, and apoptosis. [ABSTRACT FROM AUTHOR]

Since its discovery as an oncogene more than 40 years ago, Ras has been and still is in the focus of many academic and pharmaceutical labs around the world. A huge amount of work has accumulated on its biology. However, many questions about the role of the different Ras isoforms in health and disease still exist and a full understanding will require more intensive work in the future. Here we try to survey some of the structural findings in a historical perspective and how it has influenced our understanding of structure-function and mechanistic relationships of Ras and its interactions. The structures show that Ras is a stable molecular machine that uses the dynamics of its switch regions for the interaction with all regulators and effectors. This conformational flexibility has been used to create small molecule drug candidates against this important oncoprotein. [ABSTRACT FROM AUTHOR]

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described. [ABSTRACT FROM AUTHOR]

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described. [ABSTRACT FROM AUTHOR]

Paul Ebensperger and Claudia Jessen-Trefzer take up another interesting aspect of artificial protein chemistry. In this context, Wang and coworkers describe a yeast three-hybrid system to identify the protein targets of small molecules, while addressing the challenge of compound uptake into yeast cells ([8]). This Highlight Issue of I Biological Chemistry i covers various aspects of today's drug discovery process at the interface between chemistry and biology, and provides an outlook on future research directions, methods and emerging trends. [Extracted from the article]