PROteolysis TArgeting Chimeras (PROTACs) - Past, present and future.

The majority of currently used therapeutics are small molecule-based and utilize occupancy-driven pharmacology as the mode of action (MOA), in which the protein function is modulated via temporary inhibition. New modalities that operate using alternative MOAs are essential for tapping into the "undruggable" proteome. The PROteolysis Targeting Chimera (PROTAC) technology provides an attractive new approach that utilizes an event-driven MOA. Small molecule-based heterobifunctional PROTACs modulate protein target levels by hijacking the ubiquitin-proteasome system to induce degradation of the target. Here, we address important milestones in the development of the PROTAC technology, as well as emphasize key findings from this previous year and highlight future directions of this promising drug discovery modality.

Flexibility is important for inhibition of the MDM2/p53 protein-protein interaction by cyclic ß-hairpins.

Protein-protein interactions that have large, flat and featureless binding sites are difficult drug targets. In the development of their modulators conventional drug discovery strategies are often unsuccessful. Gaining a detailed understanding of the binding mode of protein-protein interaction inhibitors is therefore of vast importance for their future pharmaceutical use. The MDM2/p53 protein pair is a highly promising target for cancer treatment. Disruption of the protein complex using p53 α-helix mimetics has been shown to be a successful strategy to control p53 activity. To gain further insight into the binding of inhibitors to MDM2, the flexibility of four cyclic ß-hairpins that act as α-helical mimetics and potential MDM2/p53 interaction inhibitors was investigated in relation to their inhibitory activity. MDM2-binding of the mimetics was determined using fluorescence polarization and surface plasmon resonance assays, whereas their conformation and dynamics in solution was described by the combined experimental and computational NAMFIS analysis. Molecular flexibility was shown to be important for the activity of the cyclic ß-hairpin based MDM2 inhibitors.

Induced degradation of SNAP-fusion proteins.

Self-labeling protein tags are an efficient means to visualize, manipulate, and isolate engineered fusion proteins with suitable chemical probes. The SNAP-tag, which covalently conjugates to benzyl-guanine and -chloropyrimidine derivatives is used extensively in fluorescence microscopy, given the availability of suitable SNAP-ligand-based probes. Here, we extend the applicability of the SNAP-tag to targeted protein degradation. We developed a set of SNAP PROteolysis TArgeting Chimeras (SNAP-PROTACs), which recruit the VHL or CRBN-ubiquitin E3 ligases to induce the degradation of SNAP-fusion proteins. Endogenous tagging enabled the visualization and the selective depletion of a SNAP-clathrin light chain fusion protein using SNAP-PROTACs. The addition of PROTACs to the SNAP-tag reagent toolbox facilitates the comprehensive analysis of protein function with a single gene tagging event.

Rapid PROTAC Discovery Platform: Nanomole-Scale Array Synthesis and Direct Screening of Reaction Mixtures.

Precise length, shape, and linker attachment points are all integral components to designing efficacious proteolysis targeting chimeras (PROTACs). Due to the synthetic complexity of these heterobifunctional degraders and the difficulty of computational modeling to aid PROTAC design, the exploration of structure-activity relationships remains mostly empirical, which requires a significant investment of time and resources. To facilitate rapid hit finding, we developed capabilities for PROTAC parallel synthesis and purification by harnessing an array of preformed E3-ligand-linker intermediates. In the next iteration of this approach, we developed a rapid, nanomole-scale PROTAC synthesis methodology using amide coupling that enables direct screening of nonpurified reaction mixtures in cell-based degradation assays, as well as logD and EPSA measurements. This approach greatly expands and accelerates PROTAC SAR exploration (5 days instead of several weeks) as well as avoids laborious and solvent-demanding purification of the reaction mixtures, thus making it an economical and more sustainable methodology for PROTAC hit finding.

Retraction of "Targeted Protein Internalization and Degradation by ENDosome TArgeting Chimeras (ENDTACs)".

[This retracts the article DOI: 10.1021/acscentsci.9b00224.].

Targeted Protein Internalization and Degradation by ENDosome TArgeting Chimeras (ENDTACs).

Targeted protein degradation has generated excitement in chemical biology and drug discovery throughout academia and industry. By hijacking the machinery responsible for protein degradation via the ubiquitin proteasome system (UPS), various cellular targets have been selectively degraded. However, since the tools used, often termed PROteolysis TArgeting Chimeras (PROTACs), hijack the intracellular quality control machinery, this technology can only access targets within the cell. Extracellular targets such as growth factors, cytokines, and chemokines bind to cell surface receptors, often initiating aberrant signaling in multiple diseases such as cancer and inflammation. However, efforts to develop small molecule inhibitors for these extracellular target proteins have been challenging. Herein, we developed a proof-of-concept approach to evaluate if extracellular proteins can be internalized and degraded via the receptor-mediated endolysosomal pathway. Using a heterodimeric molecule, termed "ENDosome TArgeting Chimera" (ENDTAC), internalization and degradation of an extracellular recombinant eGFP-HT7 fusion protein was achieved by hijacking the decoy GPCR receptor, CXCR7. This proof-of-concept study suggests that using ENDTACs to co-opt the endosomal-lysosomal degradation pathway, in contrast to PROTACs using the UPS, may provide an avenue for degrading extracellular targets such as cytokines. Overall, the technology described herein provides a novel expansion to the field of targeted protein degradation.

Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction.

Small molecule nonpeptidic mimics of α-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as α-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an α-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%.