Identification of Adenosine Deaminase Inhibitors by Metal-binding Pharmacophore Screening

Abstract

Adenosine deaminase (ADA) is a human mononuclear Zn²⁺ metalloenzyme that converts adenosine to inosine. ADA is a validated drug target for cancer, but there has been little recent work on the development of new therapeutics against this enzyme. The lack of new advancements can be partially attributed to an absence of suitable assays for high-throughput screening (HTS) against ADA. To facilitate more rapid drug discovery efforts for this target, an in vitro assay was developed that utilizes the enzymatic conversion of a visibly emitting adenosine analogue to the corresponding fluorescent inosine analogue by ADA, which can be monitored via fluorescence intensity changes. Utilizing this assay, a library of ∼350 small molecules containing metal-binding pharmacophores (MBPs) was screened in an HTS format to identify new inhibitor scaffolds against ADA. This approach yielded a new metal-binding scaffold with a K_i value of 26±1 μM.

Keywords: Fluorescent Probes; Fragment Based Drug Discovery; High Throughput Screening; Medicinal Chemistry; Metalloenzymes.

Figure 1.

Top: Mechanistic depiction of ADA-mediated deamination of adenosine as well as ^tzA deamination to yield the active fluorophore ^tzI. Middle: Structure and K_i values of ADA inhibitors Pentostatin, Cladribine, and EHNA. Bottom: Crystal structure of EHNA (green) bound to the ADA active site (PDB ID: 2Z7G). Key residues, catalytic Zn²⁺ (orange sphere), and Zn²⁺-bound water (red sphere) are highlighted.

Figure 2.

Results of the MBP library screen against ADA, represented as a bar graph of percent inhibition (relative to no inhibitor) of ADA in the presence of 200 μM MBP fragment. The green dashed line denotes compounds with greater than 50% inhibition, and the red dashed line denotes compounds with greater than 75% inhibition.

Figure 3.

MBP compounds tested against ADA in this study.

Scheme 1.

General method for oxazoline synthesis by: a) cyclization reaction between ethanolamine or aminopropanol with an aromatic nitrile or b) by cyclizing amide-coupled ethanolamine. Reagents and conditions: (a) potassium acetate, hydroxylamine hydrochloride, formic acid, 100°C, 24 h, 57% yield; (b) ZnCl₂, ethanolamine, toluene, 130°C, 24 h, 75% yield; (c) ethanolamine, triethylamine, T3P, tetrahydrofuran, ethyl acetate, 70°C, 16 h, 24% yield; (d) EDC, HOBt, triethylamine, ethanolamine, DMF, 60°C, 16 h, 20% yield.