Affinity and cooperativity modulate ternary complex formation to drive targeted protein degradation.

Targeted protein degradation via "hijacking" of the ubiquitin-proteasome system using proteolysis targeting chimeras (PROTACs) has evolved into a novel therapeutic modality. The design of PROTACs is challenging; multiple steps involved in PROTAC-induced degradation make it difficult to establish coherent structure-activity relationships. Herein, we characterize PROTAC-mediated ternary complex formation and degradation by employing von Hippel-Lindau protein (VHL) recruiting PROTACs for two different target proteins, SMARCA2 and BRD4. Ternary-complex attributes and degradation activity parameters are evaluated by varying components of the PROTAC's architecture. Ternary complex binding affinity and cooperativity correlates well with degradation potency and initial rates of degradation. Additionally, we develop a ternary-complex structure modeling workflow to calculate the total buried surface area at the interface, which is in agreement with the measured ternary complex binding affinity. Our findings establish a predictive framework to guide the design of potent degraders.

Profiling of diverse tumor types establishes the broad utility of VHL-based ProTaCs and triages candidate ubiquitin ligases.

The success of small molecule therapeutics that promotes degradation of critical cancer targets has fueled an intense effort to mimic this activity with bispecific molecules called PROTACs (proteolysis targeting chimeras). The simultaneous binding of PROTACs to a ligase and target can induce proximity-driven ubiquitination and degradation. VHL and CRBN are the two best characterized PROTAC ligases, but the rules governing their cellular activities remain unclear. To establish these requirements and extend them to new ligases, we screened a panel of 56 cell lines with two potent PROTACs that utilized VHL, MZ1, or CRBN, dBET1 to induce degradation of BRD4. With notable exceptions, MZ1 was broadly active in the panel whereas dBET1 was frequently inactive. A search for predictive biomarkers of PROTAC activity found that expression and mutation of VHL and CRBN were themselves predictors of PROTAC activity in the cell line panel.

Harnessing the Power of Proteolysis for Targeted Protein Inactivation.

Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients.

A "Click Chemistry Platform" for the Rapid Synthesis of Bispecific Molecules for Inducing Protein Degradation.

Proteolysis targeting chimeras (PROTACs) are bispecific molecules containing a target protein binder and an ubiquitin ligase binder connected by a linker. By recruiting an ubiquitin ligase to a target protein, PROTACs promote ubiquitination and proteasomal degradation of the target protein. The generation of effective PROTACs depends on the nature of the protein/ligase ligand pair, linkage site, linker length, and linker composition, all of which have been difficult to address in a systematic way. Herein, we describe a "click chemistry" approach for the synthesis of PROTACs. We demonstrate the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel-Lindau (VHL) proteins. An AlphaScreen proximity assay was used to determine the ability of PROTACs to form the ternary ligase-PROTAC-target protein complex and a MSD assay to measure cellular degradation of the target protein promoted by PROTACs.

Loss of NAPRT1 expression by tumor-specific promoter methylation provides a novel predictive biomarker for NAMPT inhibitors.

PURPOSE: We sought to identify predictive biomarkers for a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor. EXPERIMENTAL DESIGN: We use a NAMPT inhibitor, GNE-617, to evaluate nicotinic acid rescue status in a panel of more than 400 cancer cell lines. Using correlative analysis and RNA interference (RNAi), we identify a specific biomarker for nicotinic acid rescue status. We next determine the mechanism of regulation of expression of the biomarker. Finally, we develop immunohistochemical (IHC) and DNA methylation assays and evaluate cancer tissue for prevalence of the biomarker across indications. RESULTS: Nicotinate phosphoribosyltransferase (NAPRT1) is necessary for nicotinic acid rescue and its expression is the major determinant of rescue status. We demonstrate that NAPRT1 promoter methylation accounts for NAPRT1 deficiency in cancer cells, and NAPRT1 methylation is predictive of rescue status in cancer cell lines. Bisulfite next-generation sequencing mapping of the NAPRT1 promoter identified tumor-specific sites of NAPRT1 DNA methylation and enabled the development of a quantitative methylation-specific PCR (QMSP) assay suitable for use on archival formalin-fixed paraffin-embedded tumor tissue. CONCLUSIONS: Tumor-specific promoter hypermethylation of NAPRT1 inactivates one of two NAD salvage pathways, resulting in synthetic lethality with the coadministration of a NAMPT inhibitor. NAPRT1 expression is lost due to promoter hypermethylation in most cancer types evaluated at frequencies ranging from 5% to 65%. NAPRT1-specific immunohistochemical or DNA methylation assays can be used on archival formalin paraffin-embedded cancer tissue to identify patients likely to benefit from coadministration of a Nampt inhibitor and nicotinic acid.

The acidic tail of the Cdc34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCdc4.

Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCF(Cdc4), functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF(Cdc4) and makes a major contribution to the submicromolar K(m) of Cdc34 for SCF(Cdc4). Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly.

Dbf2-Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis.

Exit from mitosis is characterized by a precipitous decline in cyclin-dependent kinase (Cdk) activity, dissolution of mitotic structures, and cytokinesis. In Saccharomyces cerevisiae, mitotic exit is driven by a protein phosphatase, Cdc14, which is in part responsible for counteracting Cdk activity. Throughout interphase, Cdc14 is sequestered in the nucleolus, but successful anaphase activates the mitotic exit network (MEN), which triggers dispersal of Cdc14 throughout the cell by a mechanism that has remained unknown. In this study, we show that a MEN component, protein kinase Dbf2-Mob1, promotes transfer of Cdc14 to the cytoplasm and consequent exit from mitosis by direct phosphorylation of Cdc14 on serine and threonine residues adjacent to a nuclear localization signal (NLS), thereby abrogating its NLS activity. Our results define a mechanism by which the MEN promotes exit from mitosis.