Developing Inhibitors of the p47phox–p22phox Protein–Protein Interaction by Fragment-Based Drug Discovery

Abstract

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Targeting the Redox Landscape in Cancer Therapy

“…For instance, the natural organic compound apocynin inhibits p47phox membrane translocation, and thus activates NOX2, but it also inhibits Rho kinases, thus leading to cell cycle arrest [69,70,108,163]. In a recent study, Solbak et al used fragment-based drug discovery to develop dimeric NOX2 inhibitors that target p47phox-p22phox protein-protein interaction [164]. The pan-NOX inhibitor VAS2870 interferes with NOX binding proteins, and hence inhibits NOX complex formation [54,165,166].…”

Identification of Novel Fragments Binding to the PDZ1‐2 Domain of PSD‐95

“…To further characterize the 63 primary FP hits and exclude other types of false positives, such as aggregation‐based promiscuous inhibitors, [18] we conducted a series of SPR validation experiments. SPR is a sensitive technique that can detect direct binding to the protein and unusual sensorgram shapes or response levels above those of comparative control compounds are indicative of false positives [17,19] . In SPR, the fragments were injected in a OneStep concentration gradient at 0.5 and 1 mM (4 % DMSO) over immobilized PSD‐95 PDZ1‐2 in a 384‐well plate format.…”

“…The affinity of 4 has previously been determined to be 10 nM by ITC ( K d ) and FP ( K i ); [4a,20] here a K d of 21–73 nM was determined by SPR (Figure S2). Thereby, the PDZ1‐2 domain binding pocket is blocked by 4 during the counter‐test and fragment binding to the site is hindered leading to reduced response [19,21] . Out of the 63 primary FP hits, 28 were considered SPR validated as they gave a MW normalized response level higher than 40 % of the response level of the weak control compound 2 in the primary SPR experiment ( 2 and the hits showed response levels around 10 RU and 4–20 RU, respectively).…”

“…SPR is a sensitive technique that can detect direct binding to the protein and unusual sensorgram shapes or response levels above those of comparative control compounds are indicative of false positives. [17,19] In SPR, the fragments were injected in a OneStep concentration gradient at 0.5 and 1 mM (4 % DMSO) over immobilized PSD-95 PDZ1-2 in a 384-well plate format. An octamer GluN2B-derived peptide (LSSIESDV-OH, 3 [14] ) and 2 were used as a positive controls ( Figure S2).…”

“…Thereby, the PDZ1-2 domain binding pocket is blocked by 4 during the counter-test and fragment binding to the site is hindered leading to reduced response. [19,21] Out of the 63 primary FP hits, 28 were considered SPR validated as they gave a MW normalized response level higher than 40 % of the response level of the weak control compound 2 in the primary SPR experiment (2 and the hits showed response levels around 10 RU and 4-20 RU, respectively). Also, these fragments had an "S" value � 0.3, where S is a calculated metric based on the responses from the primary test and the counter-test [21] (S = 0.3 corresponds to a reduction in response level of 50 %).…”