Fluorescing Isofunctional Ribonucleosides: Assessing Adenosine Deaminase Activity and Inhibition

Abstract

The enzymatic conversion of isothiazolo[4,3‐d]pyrimidine‐based adenosine (tzA) and 2‐aminoadenosine (tz2‐AA) analogues to the corresponding isothiazolo[4,3‐d]pyrimidine‐based inosine (tzI) and guanosine (tzG) derivatives is evaluated and compared to the conversion of native adenosine to inosine. Henri–Michaelis–Menten analyses provides the foundation for a high‐throughput screening assay, and the efficacy of the assay is showcased by fluorescence‐based analysis of tzA conversion to tzI in the presence of known and newly synthesized inhibitors.

Identification of Adenosine Deaminase Inhibitors by Metal‐binding Pharmacophore Screening

“…Taking advantage of the emissive properties of tz A and its similar reaction time to adenosine, we were able to translate this enzymatic assay to a more useful HTS format using standard plate readers. To validate the HTS format and methods used, EHNA, a known ADA inhibitor, was assessed and found to have an IC 50 =6 nM, consistent with previously reported values (Figure S1) [1a,7d,e] . One of the more potent fragments identified in the screen, 3 was found to have an IC 50 =93 μM, providing an excellent starting point for additional study (Figure S3).…”

“…Fluorescence changes were monitored following excitation at 322 nm and an emission wavelength of 410 nm. As a positive control, EHNA was screened in a 10‐point inhibition curve (see Experimental part and SI; Figure S1), and was determined to have an IC 50 value of 6±2 nM, consistent with literature values [1a,7d,e] . All MBP fragments were screened in triplicate with a positive (EHNA 10 μM) and negative (buffer only) control.…”

“…A library of ∼350 MBPs was screened at an initial concentration of 200 μM in an HTS format, based on the tz A to tz I reaction [7e] in a 96‐well plate format. Fluorescence changes were monitored following excitation at 322 nm and an emission wavelength of 410 nm.…”

“…The key to this screening campaign was the use of an emissive adenosine surrogate ( tz A, Figure 1), which is effectively deaminated by ADA to the corresponding emissive inosine derivative [8] . tz A was previously found to have a similar K M and k cat to native adenosine [7e] . Its conversion to tz I can be observed by fluorescence upon excitation in the near UV wavelength range, a range typically outside the absorption spectra of the native nucleosides and simple pharmacophores.…”

“…This MBP approach is highly compatible with fragment‐based drug discovery (FBDD). Herein, we utilize the previously described tz A to tz I biophysical assay [7e] in an HTS format, and use this tool to evaluate a library of ∼350 MBPs in an effort to discover new, metal‐binding inhibitors of ADA. Subsequent lead elaboration, as guided by structure‐activity relationship (SAR) analysis, resulted in the identification of structural motifs that contributed to fragment activity, resulting in the discovery of a new imidazoline scaffold for ADA inhibitors.…”