László Radnai

Proximity labeling typically identifies interactomes of single protein or RNA baits, limiting its use for mechanisms distributed across molecular systems. Here, we establish MesoMap, a chromatin-focused mesoscale proximity-labeling strategy that installs nanoscale photocatalysts on multiple histone baits to monitor drug-induced redistribution of proteins near chromatin. MesoMap captured HDAC inhibitor-induced remodeling of chromatin-associated proteins and, in a proof-of-concept de-orphaning application, prioritized a chromatin-proximal CLK/splicing axis for SR-1815, a phenotypically discovered SynGAP-restoring small molecule. Integration with kinase assays, splicing analysis, inhibitor comparisons, and published neuronal validation supports a model in which SR-1815 regulates SynGAP abundance through polypharmacological kinase modulation, including CLK-dependent effects on splicing. Together, these studies establish MesoMap as a complementary approach for resolving drug-induced protein redistribution near chromatin and accelerating mechanism-of-action studies for phenotypic chemical probes.

Traditional phenotypic drug discovery platforms suffer from poor scalability and/or a lack of mechanistic understanding of discovered probes. We address this by creating Endo-GeneScreen (EGS), a high-throughput platform that identifies small molecules that regulate endogenous protein levels encoded by a preselected target gene within disease-modeling contexts. Two initial screens identify >40 validated small molecules that boost endogenous neuronal Syngap1 levels, a gene that causes a neurodevelopmental disorder when haploinsufficient. EGS assays also accelerate preclinical development of drug candidates and facilitate mode-of-action deconvolution studies of orphaned probes. SR-1815 represents a fully validated proof-of-concept candidate from the platform. It is a previously unknown drug-like small molecule multikinase inhibitor that regulates splicing of Syngap1 transcripts. It restores SynGAP protein abundance to wildtype levels and mitigates major cellular consequences of Syngap1 loss-of-function. Thus, the EGS platform promotes identification and development of small molecules that alter the abundance of disease-linked proteins in a translationally-relevant context.

The scaffold protein Tks4 is a member of the p47^phox-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47^phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called “tandem SH3”) and a proline-rich region (PRR). In p47^phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47^phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47^phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.

SH3 domains constitute an important class of protein modules involved in a variety of cellular functions. They participate in protein-protein interactions via their canonical ligand binding interfaces composed of several evolutionarily conserved aromatic residues forming binding grooves for typical (PxxP) and atypical (PxxxPR, RxxK, RKxxY) binding motifs. The calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1, or Caskin1, a multidomain scaffold protein regulating the cortical actin filaments, is enriched in neural synapses in mammals. Based on its known interaction partners and knock-out animal studies, Caskin1 may play various roles in neural function and it is thought to participate in several pathological processes of the brain. Caskin1 has a single, atypical SH3 domain in which key aromatic residues are missing from the canonical binding groove. No protein interacting partner for this SH3 domain has been identified yet. Nevertheless, we have recently demonstrated the specific binding of this SH3 domain to the signaling lipid mediator lysophospatidic acid (LPA) in vitro. Here we report the solution NMR structure of the human Caskin1 SH3 domain and analyze its structural features in comparison with other SH3 domains exemplifying different strategies in target selectivity. The key differences revealed by our structural study show that the canonical binding groove found in typical SH3 domains accommodating proline-rich motifs is missing in Caskin1 SH3, most likely excluding a bona fide protein target for the domain. The LPA binding site is distinct from the altered protein binding groove. We conclude that the SH3 domain of Caskin1 might mediate the association of Caskin1 with membrane surfaces with locally elevated LPA content.

Scaffold proteins are typically thought of as multi-domain “bridging molecules.” They serve as crucial regulators of key signaling events by simultaneously binding multiple participants involved in specific signaling pathways. In the case of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) binding, the activated EGFR contacts cytosolic SRC tyrosine-kinase, which then becomes activated. This process leads to the phosphorylation of SRC-substrates, including the tyrosine kinase substrates (TKS) scaffold proteins. The TKS proteins serve as a platform for the recruitment of key players in EGFR signal transduction, promoting cell spreading and migration. The TKS4 and the TKS5 scaffold proteins are tyrosine kinase substrates with four or five SH3 domains, respectively. Their structural features allow them to recruit and bind a variety of signaling proteins and to anchor them to the cytoplasmic surface of the cell membrane. Until recently, TKS4 and TKS5 had been recognized for their involvement in cellular motility, reactive oxygen species-dependent processes, and embryonic development, among others. However, a number of novel functions have been discovered for these molecules in recent years. In this review, we attempt to cover the diverse nature of the TKS molecules by discussing their structure, regulation by SRC kinase, relevant signaling pathways, and interaction partners, as well as their involvement in cellular processes, including migration, invasion, differentiation, and adipose tissue and bone homeostasis. We also describe related pathologies and the established mouse models.

Myosin IIs, actin-based motors that utilize the chemical energy of ATP to generate force, have potential as therapeutic targets. Their heavy chains differentiate the family into muscle (skeletal [SkMII], cardiac, smooth) and nonmuscle myosin IIs. Despite therapeutic potential for muscle disorders, no SkMII-specific inhibitor has been reported and characterized. Here we present the discovery, synthesis and characterization of “skeletostatins”, novel derivatives of the pan-myosin II inhibitor blebbistatin, with selectivity within the myosin IIs for SkMII. In addition, the skeletostatins bear improved potency, solubility and photostability, without cytotoxicity. Based on its optimal in vitro profile, Skeletostatin 1’s in vivo tolerability, efficacy and pharmacokinetics were determined. Skeletostatin 1 was well-tolerated in mice, impaired motor performance, and had an excellent muscle to plasma ratio. Skeletostatins are useful probes for basic research and a strong starting point for drug development.

Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we have developed a cell-based assay for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 hours. Living cells, nuclei and nuclei of dead cells are revealed by a single staining step with three fluorescent dyes, followed by live cell imaging using an automated fluorescence microscope. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio is expected to increase over time, as the ratio of multinucleated cells increases in the population. Cytotoxicity is quantified as the ratio of dead nuclei to total nuclei. A screening window coefficient (Z’) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC₅₀values for four compounds can be reliably determined in a single 96-well plate by using only six different compound concentrations. An excellent test-retest reliability (R²=0.998) was found for EC₅₀values covering a ∼1500-fold range of potencies. The robustness, simplicity and flexibility of the assay is demonstrated here by using modulators of actin dynamics with different mechanisms of action (jasplakinolide, cytochalasin D and swinholide A) and derivatives of the nonmuscle myosin II inhibitor blebbistatin.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

The non-receptor tyrosine kinase Src is a central component of the EGF signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues: 466-474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues: 508-511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to synergistic enhancement of binding strength and a highly stable, “bidentate”-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least three hours in living cells. We conclude that the interaction of Tks4 with Src may result in the long-term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation.