Development of a PROTAC Targeting Chk1.

A series of Chk1 degraders were designed and synthesized. The degraders were developed through the conjugation of a promiscuous kinase binder and thalidomide. One of the degraders PROTAC-2 was able to decrease Chk1 levels in a concentration-dependent manner in A375 cells.

Selective Smurf1 E3 ligase inhibitors that prevent transthiolation.

Smurf1 is a HECT E3 ligase that is genetically micro-duplicated in human patients and is associated with osteoporosis. Smurf1 mice on the other hand show an increase in bone density as they age, while being viable and fertile. Therefore, Smurf1 is a promising drug target to treat osteoporosis.

Inhibiting protein synthesis to treat malaria.

Covalent prodrugs inhibit protein synthesis targets killing parasites but not human cells.

Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains that allows consideration of their dynamic disorder, and used it to analyze a set of D-motif peptide-mitogen-activated protein kinase (MAPK) associations to validate the approach and define unknown binding structures.

Methodology for Identification of Cysteine-Reactive Covalent Inhibitors.

Covalent fragments are an emerging technology to discover covalent ligands in target-based or phenotypic screens. Here we describe screening of cysteine-reactive covalent fragments against a protein of interest using mass spectrometry or enzymatic methods.

Discovery of covalent enzyme inhibitors using virtual docking of covalent fragments.

Here we present a virtual docking screen of 1648 commercially available covalent fragments, and identified covalent inhibitors of cysteine protease cathepsin L. These inhibitors did not inhibit closely related protease cathepsin B. Thus, we have established virtual docking of covalent fragments as an approach to discover covalent enzyme inhibitors.

Monitoring PARKIN RBR Ubiquitin Ligase Activation States with UbFluor.

PARKIN is a RING-Between-RING (RBR) E3 ligase, which ubiquitinates mitochondrial proteins in response to mitochondrial damage. Ser of PARKIN is phosphorylated by kinase PINK1 (pPARKIN), which causes partial PARKIN activation. PINK1 also phosphorylates Ser of ubiquitin (pUb), which further activates pPARKIN.

Structural Basis of Substrate Recognition and Covalent Inhibition of Cdu1 from Chlamydia trachomatis.

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionarily related protease adenain, a target-hopping screening approach on a focused set of adenain inhibitors was investigated. The cyanopyrimidine-based inhibitors identified represent the first active-site-directed small-molecule inhibitors of Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyanopyrimidines, as well as with its substrate ubiquitin, were obtained.

Dynamic recruitment of ubiquitin to mutant huntingtin inclusion bodies.

Many neurodegenerative diseases, such as Huntington's disease, are hallmarked by the formation of intracellular inclusion bodies (IBs) that are decorated with ubiquitin, proteasomes and chaperones. The apparent enrichment of ubiquitin and components involved in protein quality control at IBs suggests local ubiquitin-dependent enzymatic activity. In this study, we examine recruitment of ubiquitin to IBs of polyglutamine-expanded huntingtin fragments (mHtt) by using synthesized TAMRA-labeled ubiquitin moieties.

High-Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor.

HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases.

UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN.

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes.

Dissecting the Specificity of Adenosyl Sulfamate Inhibitors Targeting the Ubiquitin-Activating Enzyme.

Targeting the activating enzymes (E1) of ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) has emerged as a promising anti-cancer strategy, possibly overcoming the ineffectiveness of proteasome inhibitors against solid tumors. Here, we report crystal structures of the yeast ubiquitin E1 (Uba1) with three adenosyl sulfamate inhibitors exhibiting different E1 specificities, which are all covalently linked to ubiquitin. The structures illustrate how the chemically diverse inhibitors are accommodated within the adenylation active site.

UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis.

HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s.

UbFluor: A Mechanism-Based Probe for HECT E3 Ligases.

Homologous to E6AP Carboxyl Terminus E3 ubiquitin ligases (HECT, ~28 known) are genetically implicated in cancer, neurological, hypertensive, and autoimmune disorders, and are potential drug targets to treat these diseases. The major bottleneck in the field of HECT E3s is a lack of simple assays to quantify the enzymatic activity of these enzymes in the presence of small molecules. Typical assays require E1, E2, HECT E3, ubiquitin (Ub), ATP and additional reagents to detect the resulting free poly-ubiquitin chains.

Covalent Tethering of Fragments For Covalent Probe Discovery.

Covalent probes and drugs have found widespread use as research tools and clinical agents. Covalent probes are useful because of their increased intracellular potency and because covalent labeling of cellular proteins can be tracked using click chemistry. Covalent drugs, on the other hand, can overcome drug resistance toward their reversible counterparts.

Facile synthesis of covalent probes to capture enzymatic intermediates during E1 enzyme catalysis.

We report a facile synthetic strategy to prepare UBL-AMP electrophilic probes that form a covalent bond with the catalytic cysteine of cognate E1s, mimicking the tetrahedral intermediate of the E1-UBL-AMP complex. These probes enable the structural and biochemical study of both canonical- and non-canonical E1s.

A Small Molecule That Switches a Ubiquitin Ligase From a Processive to a Distributive Enzymatic Mechanism.

E3 ligases are genetically implicated in many human diseases, yet E3 enzyme mechanisms are not fully understood, and there is a strong need for pharmacological probes of E3s. We report the discovery that the HECT E3 Nedd4-1 is a processive enzyme and that disruption of its processivity by biochemical mutations or small molecules switches Nedd4-1 from a processive to a distributive mechanism of polyubiquitin chain synthesis. Furthermore, we discovered and structurally characterized the first covalent inhibitor of Nedd4-1, which switches Nedd4-1 from a processive to a distributive mechanism.

Identification of non-peptidic cysteine reactive fragments as inhibitors of cysteine protease rhodesain.

Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of T. brucei.

An inhibitor of ubiquitin conjugation and aggresome formation.

Proteasome inhibitors have revolutionized the treatment of multiple myeloma, and validated the therapeutic potential of the ubiquitin proteasome system (UPS). It is believed that in part, proteasome inhibitors elicit their therapeutic effect by inhibiting the degradation of misfolded proteins, which is proteotoxic and causes cell death. In spite of these successes, proteasome inhibitors are not effective against solid tumors, thus necessitating the need to explore alternative approaches.

Catalytically Important Residues of E6AP Ubiquitin Ligase Identified Using Acid-Cleavable Photo-Cross-Linkers.

Inactivation of the E6AP E3 ubiquitin ligase (UBE3A gene) causes Angelman syndrome, while aberrant degradation of p53 by E6AP is implicated in cervical cancers. Herein, we describe the development of photo-cross-linkers to discover catalytic residues of E6AP. Using these cross-linkers, we identified covalent modifications of the E6AP catalytic cysteine and two lysines: Lys(847) and Lys(799).

Protein ubiquitination and formation of polyubiquitin chains without ATP, E1 and E2 enzymes.

Studying protein ubiquitination is difficult due to the complexity of the E1-E2-E3 ubiquitination cascade. Here we report the discovery that C-terminal ubiquitin thioesters can undergo direct transthiolation with the catalytic cysteine of the model HECT E3 ubiquitin ligase Rsp5 to form a catalytically active Rsp5∼ubiquitin thioester (Rsp5∼Ub). The resulting Rsp5∼Ub undergoes efficient autoubiquitination, ubiquitinates protein substrates, and synthesizes polyubiquitin chains with native Ub isopeptide linkage specificity.

A fragment-based method to discover irreversible covalent inhibitors of cysteine proteases.

A novel fragment-based drug discovery approach is reported which irreversibly tethers drug-like fragments to catalytic cysteines. We attached an electrophile to 100 fragments without significant alterations in the reactivity of the electrophile. A mass spectrometry assay discovered three nonpeptidic inhibitors of the cysteine protease papain.

Crosstalk between kinases and Nedd4 family ubiquitin ligases.

A dazzling array of human biological processes achieves coordination and balance through the posttranslational modification of protein residues with phosphate (95 Da) or ubiquitin (8565 Da). Over the past years, a reciprocal communication has become recognized between phosphorylating (kinases) and ubiquitinating (E3 ligases) enzymes. Such crosstalk occurs when a kinase acts on a ligase or vice versa to modify the catalytic activity, substrate specificity, or subcellular localization of the modified enzyme.