Large-scale chemoproteomics expedites ligand discovery and predicts ligand behavior in cells.

Chemical modulation of proteins enables a mechanistic understanding of biology and represents the foundation of most therapeutics. However, despite decades of research, 80% of the human proteome lacks functional ligands. Chemical proteomics has advanced fragment-based ligand discovery toward cellular systems, but throughput limitations have stymied the scalable identification of fragment-protein interactions. We report proteome-wide maps of protein-binding propensity for 407 structurally diverse small-molecule fragments. We verified that identified interactions can be advanced to active chemical probes of E3 ubiquitin ligases, transporters, and kinases. Integrating machine learning binary classifiers further enabled interpretable predictions of fragment behavior in cells. The resulting resource of fragment-protein interactions and predictive models will help to elucidate principles of molecular recognition and expedite ligand discovery efforts for hitherto undrugged proteins.

Cobalamins Function as Allosteric Activators of an Angelman Syndrome-Associated UBE3A/E6AP Variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

Discovery of Molecular Glue Degraders via Isogenic Morphological Profiling.

Molecular glue degraders (MGDs) are small molecules that degrade proteins of interest via the ubiquitin-proteasome system. While MGDs were historically discovered serendipitously, approaches for MGD discovery now include cell-viability-based drug screens or data mining of public transcriptomics and drug response datasets. These approaches, however, have target spaces restricted to the essential proteins. Here we develop a high-throughput workflow for MGD discovery that also reaches the nonessential proteome. This workflow begins with the rapid synthesis of a compound library by sulfur(VI) fluoride exchange chemistry coupled to a morphological profiling assay in isogenic cell lines that vary in levels of the E3 ligase CRBN. By comparing the morphological changes induced by compound treatment across the isogenic cell lines, we were able to identify FL2-14 as a CRBN-dependent MGD targeting the nonessential protein GSPT2. We envision that this workflow would contribute to the discovery and characterization of MGDs that target a wider range of proteins.

Paralog-dependent isogenic cell assay cascade generates highly selective SLC16A3 inhibitors.

Despite being considered druggable and attractive therapeutic targets, most of the solute carrier (SLC) membrane transporters remain pharmacologically underexploited. One of the reasons for this is a lack of reliable chemical screening assays, made difficult by functional redundancies among SLCs. In this study we leveraged synthetic lethality between the lactate transporters SLC16A1 and SLC16A3 in a screening strategy that we call paralog-dependent isogenic cell assay (PARADISO). The system involves five isogenic cell lines, each dependent on various paralog genes for survival/fitness, arranged in a screening cascade tuned for the identification of SLC16A3 inhibitors. We screened a diversity-oriented library of ∼90,000 compounds and further developed our hits into slCeMM1, a paralog-selective and potent SLC16A3 inhibitor. By implementing chemoproteomics, we showed that slCeMM1 is selective also at the proteome-wide level, thus fulfilling an important criterion for chemical probes. This study represents a framework for the development of specific cell-based drug discovery assays.

FAT10 and NUB1L cooperate to activate the 26S proteasome.

The interaction of the 19S regulatory particle of the 26S proteasome with ubiquitylated proteins leads to gate opening of the 20S core particle and increases its proteolytic activity by binding of the ubiquitin chain to the inhibitory deubiquitylation enzyme USP14 on the 19S regulatory subunit RPN1. Covalent modification of proteins with the cytokine inducible ubiquitin-like modifier FAT10 is an alternative signal for proteasomal degradation. Here, we report that FAT10 and its interaction partner NUB1L facilitate the gate opening of the 20S proteasome in an ubiquitin- and USP14-independent manner. We also show that FAT10 is capable to activate all peptidolytic activities of the 26S proteasome, however only together with NUB1L, by binding to the UBA domains of NUB1L and thereby interfering with NUB1L dimerization. The binding of FAT10 to NUB1L leads to an increased affinity of NUB1L for the subunit RPN1. In conclusion, the herein described cooperation of FAT10 and NUB1L is a substrate-induced mechanism to activate the 26S proteasome.

Identification of Small-Molecule Activators of the Ubiquitin Ligase E6AP/UBE3A and Angelman Syndrome-Derived E6AP/UBE3A Variants.

Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10% of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical cross-linking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment.

Fluorescently Labelled ATP Analogues for Direct Monitoring of Ubiquitin Activation.

Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear - in contrast to earlier reports - only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.

Formation of the Alarmones Diadenosine Triphosphate and Tetraphosphate by Ubiquitin- and Ubiquitin-like-Activating Enzymes.

Diadenosine polyphosphates (ApnAs) such as diadenosine tri- and tetraphosphates are formed in prokaryotic as well as eukaryotic cells. Since upon stress intracellular ApnA concentrations increase, it was postulated that ApnAs are alarmones triggering stress-adaptive processes. The major synthesis pathway of ApnAs is assumed to be a side reaction of amino acid activation. How this process is linked to stress adaptation remains enigmatic. The first step of one of the most prominent eukaryotic post-translational modification systems-the conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubl) to target proteins-involves the formation of an adenylate as intermediate. Like ApnA formation, Ub and Ubl conjugation is significantly enhanced during stress conditions. Here, we demonstrate that diadenosine tri- and tetraphosphates are indeed synthesized during activation of Ub and Ubls. This links one of the most prevalent eukaryotic protein-modification systems to ApnA formation for the first time.

Structural dynamics of the E6AP/UBE3A-E6-p53 enzyme-substrate complex.

Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 enzyme-substrate complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 enzyme-substrate complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53.

A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome.

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.