The synthesis of a number of N-glycosyl-N-alkyl-methoxyamine bifunctional linkers is described. The linkers contain an N-methoxyamine functional group for conjugation to carbohydrates and a terminal group, such as an amine, azide, thiol, or carboxylic acid, for conjugation to the probe of choice. The strategy for the linker synthesis is rapid (3-4 steps) and efficient (51-96% overall yield), and many of the linkers can be synthesized using a three-step one-pot strategy. Moreover, the linkers can be conjugated to glycans in excellent yield and they show excellent stability toward hydrolytic cleavage.
Lactate dehydrogenases
(LDHs) are tetrameric enzymes of major significance
in cancer metabolism as well as promising targets for cancer therapy.
However, their wide and polar catalytic sites make them a challenging
target for orthosteric inhibition. In this work, we conceived to target
LDH tetramerization sites with the ambition of disrupting their oligomeric
state. To do so, we designed a protein model of a dimeric LDH-H. We
exploited this model through WaterLOGSY nuclear magnetic resonance
and microscale thermophoresis for the identification and characterization
of a set of α-helical peptides and stapled derivatives that
specifically targeted the LDH tetramerization sites. This strategy
resulted in the design of a macrocyclic peptide that competes with
the LDH tetramerization domain, thus disrupting and destabilizing
LDH tetramers. These peptides and macrocycles, along with the dimeric
model of LDH-H, constitute promising pharmacological tools for the de novo design and identification of LDH tetramerization
disruptors. Overall, our study demonstrates that disrupting LDH oligomerization
state by targeting their tetramerization sites is achievable and paves
the way toward LDH inhibition through this novel molecular mechanism.
Metalloenzyme arginase is a therapeutically relevant target associated with tumor growth. To fight cancer immunosuppression, arginase activity can be modulated by small chemical inhibitors binding to its catalytic center. To better understand molecular mechanisms of arginase inhibition, a careful computer-aided mechanistic structural investigation of this enzyme was conducted. Using molecular dynamics (MD) simulations in the microsecond range, key regions of the protein active site were identified and their flexibility was evaluated and compared. A cavity opening phenomenon was observed, involving three loops directly interacting with all known ligands, while metal coordinating regions remained motionless. A novel dynamic 3D pharmacophore analysis method termed dynophores has been developed that allows for the construction of a single 3D-model comprising all ligand-enzyme interactions occurring throughout a complete MD trajectory. This new technique for the in silico study of intermolecular interactions allows for loop flexibility analysis coupled with movements and conformational changes of bound ligands. Presented MD studies highlight the plasticity of the size of the arginase active site, leading to the hypothesis that larger ligands can enter the cavity of arginase. Experimental testing of a targeted fragment library substituted by different aliphatic groups validates this hypothesis, paving the way for the design of arginase inhibitors with novel binding patterns.
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