Abstract
Human arylamine N-acetyltransferase 1 (hNAT1) has become an attractive potential biomarker for estrogen-receptor-positive breast cancers. We describe here the mechanism of action of a selective non-covalent colorimetric biosensor for the recognition of hNAT1 and its murine homologue, mNat2, over their respective isoenzymes, leading to new opportunities in diagnosis. On interaction with the enzyme, the naphthoquinone probe undergoes an instantaneous and striking visible color change from red to blue. Spectroscopic, chemical, molecular modelling and biochemical studies reported here show that the color change is mediated by selective recognition between the conjugate base of the sulfonamide group within the probe and the conjugate acid of the arginine residue within the active site of both hNAT1 and mNat2. This represents a new mechanism for selective biomarker sensing and may be exploited as a general approach to the specific detection of biomarkers in disease.
Highlights
Diagnosis of breast cancer combines non-invasive examinations, such as mammography, ultrasound or magnetic resonance imaging and biopsy tests
Proteomic and microarray analyses have identified the overexpression of human arylamine N-acetyltransferase 1 in estrogenreceptor-positive ductal and lobular breast cancers [8,9,10] and more recently in male breast cancers [11]; this overexpression inversely correlates to tumor grade [12]
We describe a set of spectroscopic, chemical, molecular modeling and biochemical studies to interrogate the key molecular interactions between human arylamine N-acetyltransferase 1 (hNAT1) or its murine homologue, mNat2, and naphthoquinone 1 which lead to the observed color change event
Summary
Diagnosis of breast cancer combines non-invasive examinations, such as mammography, ultrasound or magnetic resonance imaging and biopsy tests. An instantaneous distinctive color change from red to blue is observed upon binding of these naphthoquinone ligands, such as compound 1, to both hNAT1 and mNat, which share more than 80% identity in amino acid sequence and are functionally homologous (Figure 1) [16,17]. No such shift in the lmax of naphthoquinone 1 was observed in the presence of the other human and murine NAT enzymes despite the high number of identical residues (.70%) (Figure 1), nor with NATs from prokaryotes [16]. Since hNAT1 is a candidate biomarker in breast cancer, it was reasoned that understanding the mechanism of recognition and color change between this family of naphthoquinone probes and hNAT1 could allow both the development of these probes for tumor subtype diagnosis and the application of this technology to other protein families
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