Fragment-Based Identification of a Locus in the Sec7 Domain of Arno for the Design of Protein–Protein Interaction Inhibitors

Abstract

By virtual screening using a fragment-based drug design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno, and then their ability to interfere with the Arno-catalyzed nucleotide exchange on the G-protein Arf1 was evaluated. By use of SPR, NMR, and fluorescence assays, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated. Then the binding mode of one fragment and of a more active analogue was solved by X-ray crystallography. This highlighted the role of stable and transient pockets at the Sec7 domain surface in the discovery and binding of interfering compounds. These results provide structural information on how small organic compounds can interfere with the Arf1-Arno Sec7 domain interaction and may guide the rational drug design of competitive inhibitors of Arno enzymatic activity.

Targeting the Small GTPase Superfamily through Their Regulatory Proteins

“…Rouhana et al [123] conducted an in silico fragment screening to identify compounds that bind to the catalytic Sec7 GEF domain of ARNO.3 3f ragments were predicted to bind within small pockets near predicted hotspots.T he potencyof the compounds was evaluated using afluorescent nucleotideexchange assay,N MR spectroscopy,a nd SPR assay.C ompounds that aggregated were removed, and PA INs motifs were considered. X-ray crystal structures were solved for three of the compounds; 69 (Figure 21 A, K iapp = 3.7 mm) bound in the Sec7 domain near key residues required to form interactions in the Arf-ARNO complex, thereby preventing PPIs and complex formation.…”

“…Rouhana et al . conducted an in silico fragment screening to identify compounds that bind to the catalytic Sec7 GEF domain of ARNO.…”

Targeting der kleinen GTPasen über ihre regulatorischen Proteine

“…Rouhana et al . führten ein In‐silico‐Fragmentscreening durch, um Verbindungen zu identifizieren, die an die katalytische Sec7‐GEF‐Domäne von ARNO binden.…”

Fragment-based drug discovery and protein–protein interactions

“…Primary screening techniques include protein-and ligandobserved nuclear magnetic resonance (NMR), X-ray crystallography, surface plasmon resonance, thermal shift assay (TSA), and biochemical assays. Notably, two-dimensional heteronuclear single quantum coherence (HSQC) NMR and X-ray crystallography, employed as frontline approaches at Abbott Laboratories (Abbott Park, IL, USA) and Astex Pharmaceuticals ( Cambridge, UK), respectively, deliver detailed information about fragment-binding modes at the earliest XiAP/ciAP1 primary screening and complex structure determination [62][63][64] HCv NS3 primary screening and complex structure determination 20 Bromodomain primary screening 73 and complex structure determination [71][72][73][74] Arf1-Arno complex structure determination 77 …”

“…A fragment-screening campaign was established to identify Turnbull et al fragments that interfere with the Arf1-Arno Sec7 domain interaction and inhibit GDP to GTP nucleotide exchange. 77 This represents a challenging PPI target, since the Arf1-Arno interface comprises several hot spots disseminated over a surface area close to 1500 Å 2 . First, virtual screening of approximately 3,000 "rule-of-3" compliant fragments from the ChemBridge library led to the selection of 33 fragments that could potentially interact with the hot spots (identified via in silico alanine scanning) on the Arno Sec7 domain binding surface.…”