Protein-protein interactions are a crucial element in cellular function. The wealth of information currently available on intracellular-signaling pathways has led many to appreciate the untapped pool of potential drug targets that reside downstream of the commonly targeted receptors. Over the last two decades, there has been significant interest in developing therapeutics and chemical probes that inhibit specific protein-protein interactions. Although it has been a challenge to develop small molecules that are capable of occluding the large, often relatively featureless protein-protein interaction interface, there are increasing numbers of examples of small molecules that function in this manner with reasonable potency. This article will highlight the current progress in the development of small molecule protein-protein interaction inhibitors that have applications in the treatment or study of central nervous system function and disease. In particular, we will focus upon recent work towards developing small molecule inhibitors of amyloid-beta and alpha-synuclein aggregation, inhibitors of critical components of G-protein-signaling pathways, and PDZ domain inhibitors.

Regulators of G-Protein signaling (RGS) proteins are potent negative modulators of signal transduction through G-Protein coupled receptors. They function by binding to activated (GTP-bound) Gα subunits and accelerating the rate of GTP hydrolysis. Modulation of RGS activity by small molecules is an attractive mechanism to fine-tune GPCR signaling for therapeutic and research purposes. Here we describe the pharmacologic properties and mechanism of action of CCG-50014, the most potent small molecule RGS inhibitor to date. It has an IC50 for RGS4 of 30 nM and is >20-fold selective for RGS4 over other RGS proteins. CCG-50014 binds covalently to the RGS, forming an adduct on two cysteine residues located in an allosteric regulatory site. It is not a general cysteine alkylator as it does not inhibit activity of the cysteine protease papain at concentrations >3,000 fold higher than those required to inhibit RGS4 function. It is also >1,000-fold more potent as an RGS4 inhibitor than are the cysteine alkylators N-ethylmaleimide or iodoacetamide. Analysis of the cysteine reactivity of the compound shows that compound binding to Cys107 in RGS8 inhibits Gα- binding in a manner that can be reversed by cleavage of the compound-RGS disulfide bond. If the compound reacts with Cys160 in RGS8, the adduct induces RGS denaturation and activity cannot be restored by compound removal. The high potency and good selectivity of CCG-50014 make it a useful tool for studying the functional roles of RGS4.

Regulator of G protein signaling (RGS) proteins act to temporally modulate the activity of G protein subunits after G proteincoupled receptor activation. RGS proteins exert their effect by directly binding to the activated G␣ subunit of the G protein, catalyzing the accelerated hydrolysis of GTP and returning the G protein to its inactive, heterotrimeric form. In previous studies, we have sought to inhibit this GTPase-accelerating protein activity of the RGS protein by using small molecules. In this study, we investigated the mechanism of sulfonyl]-4-nitro-benzenesulfinimidoate], a previously reported small-molecule RGS inhibitor. Here, we find that CCG-4986 inhibits RGS4 function through the covalent modification of two spatially distinct cysteine residues on RGS4. We confirm that modification of Cys132, located near the RGS/G␣ interaction surface, modestly inhibits G␣ binding and GTPase acceleration. In addition, we report that modification of Cys148, a residue located on the opposite face of RGS4, can disrupt RGS/G␣ interaction through an allosteric mechanism that almost completely inhibits the G␣-RGS protein-protein interaction. These findings demonstrate three important points: 1) the modification of the Cys148 allosteric site results in significant changes to the RGS interaction surface with G␣; 2) this identifies a "hot spot" on RGS4 for binding of small molecules and triggering an allosteric change that may be significantly more effective than targeting the actual protein-protein interaction surface; and 3) because of the modification of a positional equivalent of Cys148 in RGS8 by CCG-4986, lack of inhibition indicates that RGS proteins exhibit fundamental differences in their responses to smallmolecule ligands.

Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((, inhibit the interaction between RGS4 and G␣ o with an IC 50 value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 Ͼ 19 ϭ 16 Ͼ 8 Ͼ Ͼ 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to G␣ o . On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.