전문가기고 [Kyung Bo Kim] PROTACs: Reinvention of an old workhorse-the small molecule drug


PROTACs: Reinvention of an old workhorse-the small molecule drug

(Department of Pharmaceutical Sciences, University of Kentucky, Kyung Bo Kim, Ph.D., )


The successful completion of human genome project and proteome information has revealed a myriad of proteins that may be associated with many of the deadliest diseases, potentially offering an enormous opportunity to intervene pathological processes. However, most of these proteins are deemed as “undruggable” due to the lack of defined binding pockets that can be functionally blocked by existing drug modalities. Currently, conventional small-molecule and antibody drugs can access only about 20% of these proteins.1 As a result, expanding the landscape of druggable protein targets further into previously non-druggable disease-

promoting proteins has long been a major challenge for the pharmaceutical industry.

Developed and crafted over the past decades, a revolutionary small-molecule family called “PROTACs” could be an answer to this conundrum for pharmaceutical industry. The PROTACs (Proteolysis Targeting Chimera) work by cleverly hijacking a cell’s natural quality-control system and using it for destroying proteins of interest (Figure 1).2 Because PROTACs (“baits”) simply need binding rather than inhibiting proteins of interest (“preys”) to work, they could conceivably target non-enzymes or non-receptors: proteins that are lacking functionally relevant ligand-binding pockets and thus are largely out of reach for drug development. Another important feature of PROTACs is their sub-stoichiometric catalytic nature,3 thereby making them distinctively different from conventional drugs that are used in great stoichiometric excess of their targets to be effective. New modalities such as RNA-based silencing and gene editing can also turn-off the activity of previously unreachable protein drug targets. However, they are routinely accompanied with some inherent issues, such as delivery, storage and formulation, which make the drug development process excruciatingly long and often financially untenable. In comparison, PROTACs being small molecules in essence potentially face fewer hurdles to reaching the clinic door and also could be mass-produced, stored long in pill form. With a variety of potential protein targets for intervention, the PROTAC platform could be applied to various disease states, from eliminating hormone-dependent tumors to clearing clusters of disease-causing proteins from the brains of patients with neurological disorders including Alzheimer’s disease. The PROTAC platform is rapidly gaining momentum now that the pharmaceutical industry, attracted to the potential to intervene ~80% of the proteome untapped for drug discovery, is pouring billions into the field.

History: The creation of PROTACs

The idea to target undruggable proteins using cell’s own protein degradation machinery (UPS, Ubiquitin-Protein System) was initially brewed over chatting between Prof. Craig Crews of Yale University and Prof. Ray Deshaies of Caltech (now senior vice president of Amgen) during a Burrow Welcome Fund sponsored research retreat in 1998 at a resort on Semiahmoo Bay in northwest Washington. Started with chatting over poster sessions and continued the conversation over beers all weekend, the idea of linking E3 ligase (β-TRCP, an F-box protein) to UPS using chimeric molecules for ubiquitination and degradation of targeted proteins was soon realized by a PNAS publication by Crews’ and Deshaies’ groups.4 Because first-generation PROTACs were composed of a large phosphopeptide ligand for an E3 ubiquitin ligase (β-TRCP) on one end and a small molecule ligand that binds to proteins of interest on the other end (Figure 2a), they were only deliverable vie microneedle and also had weak activity in human cells. As a result, the concept of PROTACs was for years regarded as an academic exercise. Unfazed by initial skepticism, Crews and colleagues continuously worked to improve the PROTAC platform, eventually expanding the pool of E3 ligase to VHL (Von-Hippel Lindau) protein which recruits HIF-1α under normal oxygen concentration condition to induce ubiquitination and degradation to shut down cellular hypoxic responses.3 The replacement of β-TRCP ligand with a shorter non-phosphopeptide fragment derived from HIF-1α improved pharmaceutical properties of PROTACs to a certain degree (Figure 2b), but there remained much room for improvement.

Realizing the potential of the PROTAC platform, in 2013, Crews founded Arvinas, Inc. in New Haven, CT, USA. While the field were still confronted with some doubts, the idea to transform the PROATC concept into marketed treatment finally got a big boost: revelation of PROTAC-like mode of action for some of world’s best-selling drugs. A series of studies performed in 2010 and 2015 revealed that the multiple myeloma drugs thalidomide and lenalidomide (Revlimid®, an amino analog of thalidomide that’s been a multi-billion dollar drug for Celgene) bind to the cereblon (CRBN) E3 ligase and induce ubiquitination and degradation of transcription factors (Ikaros and Aiolos) that are critical for myeloma cell growth.5-6 The thalidomide-based protein-degrading platform was further verified by additional studies that modification of thalidomide alters the binding partners of CRBN, inducing degradation of casein kinase 1α (CK1α) and a translation termination factor GSP1 that are otherwise not targeted by CRBN.7-8 All of sudden, this has made the protein degrading platform one of the hottest new technologies for developing new drugs, opening a flood gate for new biotech businesses.

Race to the clinic: Turning PROTACs into pills

In recent years, multiple biotech firms armed with various types of protein degradation platforms have emerged with ample funding: Arvinas Inc. (New Haven, CT, USA), C4 Therapeutics (Watertown, MA, USA), Kymera (Cambridge, MA, USA), Cullgen (San Diego, CA, USA), Nurix (San Francisco, CA, USA), to name a few. Buoyed by the gold rush, big pharma companies, such as Pfizer (New York, NY, USA), Novartis (Basel, Switzerland), GlaxoSmithKline (Brentford, UK) and Amgen (Thousand Oaks, CA, USA) among others, have also launched internal efforts and forged partnerships to explore the PROTAC modality. Despite significant efforts made by the pharmaceutical industry and academia to expand the pool of E3 ligases and optimize PROTACs, only four main ones are primarily used at present. Of those, VHL and CRBN are the ones that are drawing the most attention from the drug industry in the race to get to drug market.

In collaboration with Ian Churcher at GlaxoSmithKline, Crews et al. reported the first small molecule VHL ligand (Figure 3a), preparing all small molecule PROTACs (Figure 3b).3 The small molecule VHL ligand was created by contracting and constraining the HIF-1α peptide fragment, guided by molecular dynamics and empirical SAR (Structure-Activity-Relationship) data obtained from the conventional medicinal approach. Eventually, the small molecule VHL ligand has been successfully exploited for the degradation of a number of disease-promoting proteins including hormone receptors (ER and AR), BCR-ABL, BETs, TBK1, several transmembrane receptor tyrosine kinases (EGFR, HER2, and c-Met), and TRIM.7-9

Recently, Arvinas, Inc. launched human clinical trials for small molecule VHL ligand-based PROTAC drugs, ARV-110 and ARV-471.10-11 These drugs degrade cancer-promoting proteins that are already targeted by a number of FDA-approved drugs, the androgen receptor (AR) and estrogen receptor (ER), respectively. Specifically, ARV-110 is currently being investigated for its efficacy in patients with metastatic castration-resistant prostate cancer (mCRPC) who have progressed on at least two prior systemic therapies. On the other hand, the ARV-471 clinical trial is for patients with ER(+)/HER2(-) locally advanced or metastatic breast cancer who have received prior hormonal therapy and chemotherapy. It is hoped that these once per day oral drugs may overcome limitations associated with existing drugs, by eliminating rather than inhibiting the respective hormone receptors. Phase 1 dose escalation trial data for ARV-110 and ARV-471 are expected to be available in the first and second half of 2020, respectively.

Around the same time, CRBN ligand-based PROTACs were first developed by Bradner et al,5 reporting that the thalidomide-based PROTAC can effectively degrade the cancer-promoting BRD4 via CRBN-mediated ubiquitination and degradation (Figure 4). Since then, a number of PROTACs with thalidomide analogs (more accurately the way that the phthalimide residue in thalidomide, colored circle in Figure 4a) as a CRBN-recruiting ligand have been successfully prepared to induce degradation of various proteins of disease interest: BCR-ABL, BRD9, Sirt2, CDK9, FLT3, BTK, and ALK.7, 12 Currently, C4 Therapeutics, founded on the work of James Bradner at the Dana Farber Cancer Institute, Boston, MA, USA (currently president of the Novartis Institutes for BioMedical Research), is spear-heading the effort to develop CRBN-targeted PROTAC modalities. With their drug-like chemical composition, C4 Therapeutics (MA, USA) are presently on their way to provide a clinical drug candidate using in-house Daedalus® platform (https://c4therapeutics.com/). Meanwhile, Kymera Therapeutics is developing KYM-001 which targets IRAK4, a protein involved in disease signaling pathways, which breaks down a protein linked to certain lymphomas, and expecting to begin human clinical trials soon (https://www.kymeratx.com/).

Challenges and opportunities

For many years, there has been considerable doubt about whether PROTACs can really reach to the clinic. It is largely because PROTACs shatter the Lipinski’s rule of five: chief among them is size. One of unspoken golden rules for medicinal chemists working in the lab is that a good small-molecule drug typically has to have a mass of less than 500 daltons: it has been commonly conceived that drugs smaller than 500 daltons, with sufficient oil solubility and high partition coefficient, can enter into cells. However, PROTACs are far from wieldy, ranging upwards of 1,000 daltons, yet repeatedly shown to enter cells without much difficulties. PROTACs are currently administered via various routes in mouse models or human clinical trials: intraperitoneal, subcutaneous or intravenous, and oral. It is speculated that PROTACs’ good cell permeability is because they are probably recognized by the cell membrane as two smaller molecules that happen to be tethered together, rather than a single large one.13 Perhaps, the most encouraging about the PROTAC platform is that drug developers could finally overcome the lethargic feeling about the previously conceived “undruggable” proteins, such as KRAS and MYC.14 Considering that current drug design is achieved largely via small-molecule drugs or monoclonal antibodies that bind to an active site on an enzyme or a receptor, there is no doubt that the PROTAC platform has completely reshaped the landscape of druggable proteome in drug discovery

While targeting an estimated 80% of undruggable proteins is now within reach, currently, creation of PROTACs almost exclusively rely on existing small molecule ligands that bind to druggable proteins. In this regard, the field still has not seen an evidence that a PROTAC can target and degrade a valuable undruggable protein, at least by means of publications or clinical studies. Therefore, identification of small molecules against undruggable proteins will be critical in extending the repository of PROTAC-based drugs (Figure 5). By developing a wide variety of small molecules against undruggable proteins, it is anticipated that the PROTAC platform may offer an opportunity to potentially overcome cancer resistance caused by the emergence of drug-resistant variants or post-translational modifications.

While PROTACs may naturally have off-targets due to small molecules present at both ends of PROTACs, they are so far shown to be unable to degrade off-target proteins in cell and mouse models.15 Currently, the potential side effects remain largely unknown and are expected to come to light in the future. Separately, various industry and academic teams are also working to expand the pool of E3 ligases beyond VHL and CRBN. Potentially, an increased number of E3 ligases available for PROTAC creation could enable researchers to select the best combination of E3 ligase and ligand to a protein of interest. The search for new E3 ligases is now on to fully exploit the potential of the PROTAC platform for drug development.

Human trials have just begun for the first two Arvinas drugs, so the impacts on cancer patient treatment won’t be known for several years. Two or more are also expected to enter clinical trials in the next year or so. Furthermore, the PROTAC modality is currently investigated not only for cancer but also an array of diseases, including those that affect the brain. Looking ahead, the PROTAC technology will pave the way to develop drugs that are not accessible in the past. Most importantly, the new drug development platform means a hope for patients and their family who have not benefited from existing drugs or no available therapy.


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