Structure–activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine N-acetyltransferase 1

Abstract

A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure-activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pKa, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors.

Treatment of Rats with Apocynin Has Considerable Inhibitory Effects on Arylamine N-Acetyltransferase Activity in the Liver

“…To date, only crystal structures of human NAT enzymes are available as mammalian species48; nevertheless, they have constituted highly reliable templates for inhibitor studies involving rodent NATs because of their high percentages of residue identity (>70%) (Table 1, Supplementary Figure S1)274950.…”

Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding

“…The inhibitory potency and colorimetric properties of the (HUMAN)NAT1-selective inhibitor naphthoquinone 1 were also explored in relation to alterations at positions 125, 127 and 129 using (MOUSE)NAT2_F125S, (MOUSE)NAT1*1 and (HUMAN)NAT2*4, comparing the results with those of previous studies using (HUMAN)NAT1*4, (MESAU)NAT2*1, (MOUSE)NAT2*1, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L [ 24 – 26 ] (Table 2 ). The lowest IC 50 values for naphthoquinone 1 were exhibited by (HUMAN)NAT1*4 and (MOUSE)NAT2*1 enzymes, both of which possess the triad F125, R127 and Y129.…”

“… IC 50 values were calculated using decreasing concentrations of naphthoquinone 1. NAT activity was measured following the hydrolysis rate of AcCoA (400 μM) in the presence of 4ABA for (HUMAN)NAT1*4, (MOUSE)NAT2*1 and (MESAU)NAT2*1; 5AS for (MOUSE)NAT2 mutants and (MOUSE)NAT1*1; and 2-aminofluorene for (HUMAN)NAT2*4. a adapted from [ 25 ] b adapted from [ 24 ] c adapted from [ 26 ]. Residues which are different from the corresponding residues in (HUMAN)NAT1*4 are labeled in bold.…”

“…In the present study we focused on residues 125 and 127; the role of the Y129 residue found in (HUMAN)NAT1 and (MOUSE)NAT2, at least with respect to inhibitor binding, has previously been investigated using (MESAU)NAT2, which has identical active site residues except for a leucine (L) at location 129 [ 33 ]. The results of this earlier study [ 26 ] suggest that Y129 is functionally important, at least in inhibitor recognition, and illustrate the value of (MESAU)NAT2 as a protein model for comparative studies with (HUMAN)NAT1 and (MOUSE)NAT2.…”

“…Furthermore, inhibition studies have identified a (HUMAN)NAT1/(MOUSE)NAT2 selective inhibitor, naphthoquinone 1 ( N -(3-(3,5-dimethylphenylamino)-1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzenesulphonamide; Additional file 1 : Figure S1), which undergoes a distinctive colour change from red to blue upon binding to either of these two enzymes but not to other mammalian NAT isoenzymes [ 24 , 25 ]. Virtual modelling studies [ 25 , 26 ] indicate that the interaction between naphthoquinone 1 and (HUMAN)NAT1 or (MOUSE)NAT2 depends on selective ionic interactions between the conjugate base of compound 1 and the guanidinium moiety of an active site residue, arginine 127 (R127). This is part of a group of active site residues which had previously been identified as being involved, together with phenylalanine 125 (F125) and tyrosine 129 (Y129), in substrate specificity [ 27 , 28 ].…”