Discovery of a new class of triazole based inhibitors of acetyl transferase KAT2A

Abstract We have recently developed a new synthetic methodology that provided both N-aryl-5-hydroxytriazoles and N-pyridine-4-alkyl triazoles. A selection of these products was carried through virtual screening towards targets that are contemporary and validated for drug discovery and development. This study determined a number of potential structure target dyads of which N-pyridinium-4-carboxylic-5-alkyl triazole displayed the highest score specificity towards KAT2A. Binding affinity tests of abovementioned triazole and related analogs towards KAT2A confirmed the predictions of the in-silico assay. Finally, we have run in vitro inhibition assays of selected triazoles towards KAT2A; the ensemble of binding and inhibition assays delivered pyridyl-triazoles carboxylates as the prototype of a new class of inhibitors of KAT2A.


Docking Screening
The computational analysis was launched on the AWS E2 cloud (Amazon Web Service EC2) using the following virtual centralized processing unit (VCPU): 36 RAM: 72 G HD: SSD (GP2 type) on a Scalable 2nd Gen Intel Xeon (Cascade Lake) with an all-core turbo frequency of 3.6 GHz and a single-core turbo frequency up to 3.9 GHz. Docking screening of triazoles 11-18 has been performed testing each of the candidate compounds against a precomputed database of human crystallographic protein pockets (BioGPS pocketome). 2 The BioGPS workflow used for the docking screening of triazoles 11-18 consisted of 5 parts: i) Protein refinement, achieved by using an algorithm known as "Fixpdb" that enables the preparation of the protein structure obtained from the Protein Data Bank (PDB).
Additionally, Fixpdb also processes potential solvent molecules, co-crystallised ligands, cofactors or ions to be retained for consideration in subsequent analysis, if S4 considered essential for the protein functionality. 2 ii) Cavity detection, achieved by using an algorithm known as "Flapsite" that embeds the protein structure into a tridimensional grid and allows for the identification of pocket points located only within a distance of 4 Å maximum from the closest protein atom using the GRID probe H, that discriminates according to the "shape" The 20 proteins identified by the screening have been chosen according to the best similarity score obtained for each protein pockets and the tested compounds. In particular, distribution scores of compounds 11-18 displayed a characteristic gaussian curve for each molecule that measured the probability the of these compounds to bind a protein's active site according to their S5 shape similarities. The global score of similarity values returned are represented by a number comprised between 0, meaning "no similarity", and 1, meaning "maximum similarity".

Cell Proliferation Assay (MTT Assay)
Thiazolyl blue tetrazolium bromide (MTT) assay was carried out as follows. Cells were collected and counted; 30.000 cells were seeded per well in 48-well plate. The cells were treated with compounds 16, 26c and 27a-d at different concentrations and with SAHA and then kept at 37°C in 5% CO 2 for 24 hours. At this point, 50 µL of (5 mg/mL) MTT reagent was added to each well and the plate was placed at 37°C in the incubator for 4 hours. 300 µL of dimethyl sulfoxide (DMSO) was added to each well after aspirating the supernatant. Colored formazan product was assayed spectrophotometrically at 570 nm using TECAN infinite M200 plate reader.  The fluorogenic assay for KAT2A potential inhibitors screening of compounds 16, 26c and 27ad was provided by West Bioscience protocol 10 and set up as follows:

Enhanced Cell
• A positive control, in which the potential inhibitor was excluded from the test.
• An autoacetylation control, without the presence of either the inhibitor or the H3 peptide.
• A negative control, in which the presence of the enzyme was excluded.
• And the proper test for screening the inhibition power of triazoles 16, 26c and 28a-d, with all the components present.
The inhibition test was set up as follows (Table 1) and emission = 519nm). The inhibition tests were performed, as per protocol, three times for each compound (Table 1).