Cyplecksins are covalent inhibitors of the pleckstrin homology domain of cytohesin.

Cytohesins are cytoplasmic multidomain proteins that act as guanine nucleotide exchange factors (GEFs) for small Raslike GTPases called ADP-ribosylation factors (Arfs). Their Sec7 domain catalyzes the exchange of guanosine-5’-diphosphate (GDP) for guanosine-5’-triphosphate (GTP), which activates Arf proteins like Arf1 and Arf6. Mammalian cells contain four highly homologous cytohesins (cytohesins 1–4) that are implicated in cellular processes such as b2-integrinmediated cell adhesion and actin dynamics, including Arfmediated functions, namely membrane trafficking, vesicle transport, endocytosis, and more. Moreover, cytohesin-2 (ARNO) is a cytoplasmic activator of receptor tyrosine kinase signaling by the insulin receptor (IR) and epidermal growth factor receptor (EGFR), respectively. ARNO and Arf6 also contribute to the disruptive effects of interleukin-1b (IL-1b) on endothelial stability by binding to the adaptor protein MYD88. These discoveries were greatly aided by the availability of SecinH3, an inhibitor of the cytohesin Sec7 domain. C-terminal to their Sec7 domain, cytohesins contain another functional domain called the pleckstrin homology (PH) domain (Figure 1a). Through their PH domain cytohesins are recruited to the inner leaflet of the plasma membrane and intracellular membranes by binding either to the phosphatidylinositol phosphates PIP2 or PIP3, or to activated Arf6-GTP. Cytohesin Sec7-dependent integrin activation in cell adhesion requires the presence of the PH domain, and the PH domain exerts a certain level of inhibition on the Sec7 domain that is relieved upon membrane recruitment and interaction with PIPs. These findings indicate that the functions of the cytohesin Sec7 and PH domains may be tightly interconnected. Inspired by the usefulness of SecinH3 as a chemical biology tool for elucidating previously unknown functions of cytohesin Sec7 domains, we sought to identify a small-molecule inhibitor for cytohesin PH domains. Here we report the discovery of a class of cytohesin PH domain inhibitors called Cyplecksins (Cytohesin pleckstrin homology domain inhibitors) that act by a covalent mechanism. To develop an aptamer displacement assay for HTS, we first selected an RNA aptamer that bound the PH domain of cytohesin-1. After seven selection rounds, the enriched RNA library was cloned and sequenced, and clone 6.10 was identified as a cytohesin PH domain binder (Figure S1a in the Supporting Information (SI)). Clone 6.10 bound PH domains of cytohesins 1, 2, and 3 with Kd values between 0.3 and 0.7 mm, whereas no binding to the Sec7 domain and to related PH domains could be detected (Figure S1b,c (SI)). Figure 1. Aptamer displacement assay based on RiboGreen fluorescence capture. a) Domain structure of cytohesin-2. CC: Coiled coil; PBR: polybasic region. b) The immobilized cytohesin is incubated with the aptamer and small molecules. Nonbinding molecules are removed by a buffer wash. Remaining bound aptamer is detected by RiboGreen fluorescence (bottom right); reduced fluorescence is obtained upon aptamer displacement by the small molecule (top right). c) Representative primary screening plate with a hit compound (black dot; no. 11). Positive controls (dark gray dots; nos. 87–90) lacked cytohesin-1 coating; negative controls (light gray dots; nos. 84–86) did not contain any compound. d) Chemical structures of the most active hit compounds, derivatives of 5-bromopyrimidine-2,4,6-trione.