Protac-Induced Protein Degradation in Drug Discovery: Breaking the

Perspective Cite This: J. Med. Chem. 2018, 61, 444−452

pubs.acs.org/jmc

Protac-Induced Protein Degradation in Drug Discovery: Breaking the Rules or Just Making New Ones? Ian Churcher* BenevolentBio, 40 Churchway, London NW1 1LW, U.K. ABSTRACT: Targeted protein degradation, using bifunctional small molecules (Protacs) to remove specific proteins from within cells, has emerged as a novel drug discovery strategy with the potential to offer therapeutic interventions not achievable with existing approaches. In this Perspective, the brief history of the field is surveyed from a drug discovery perspective with a focus on the key advances in knowledge which have led to the definition and exemplification of protein degradation concepts and their resulting applications to medicine discovery. The approach has the potential to bring disruptive change to drug discovery; the many potential advantages and outstanding challenges which lie ahead of this technology are discussed.

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accurately de novo predict the pharmacodynamic behavior of molecules in vivo and in the clinic. The above issues are felt most keenly when designing small molecule drugs, so increasingly, other modalities are being used to circumvent such limitations. The successful use of monoclonal antibodies and other protein agents designed to bind to specific target epitopes with high affinity and selectivity is now well established, although these agents can currently only be applied to extracellular or cell surface targets. The majority of genomic proteins and contemporary drug targets however function intracellularly and are thus only accessible to small molecule agents, though increasingly modulation of these targets can be achieved by gene silencing or other interventions at the RNA level. Although highly effective preclinically, the requisite oligonucleotide agents themselves have well documented challenges (cellular delivery, stability, biodistribution, selectivity, etc.) that must be overcome in order to develop successful medicines.3 Recent years have also seen an explosion in genome editing approaches, and this may yet hold transformational potential to correct specific genetic defects once the practicalities of convenient delivery to patients have been addressed.4 Despite the potential emergence of novel methods for modulation of intracellular targets, use of small molecules will likely retain its place in the therapeutic arsenal due to their ability to (i) access a wide range of organs and sites of action, (ii) modulate multiple targets simultaneously, and not least, (iii) be produced with relatively low cost of goods via a well understood development path. Given this, new strategies that possess these advantages of small molecules yet also move

he fundamental concepts underpinning the discovery of new drugs have remained largely unchanged for around 100 years. Since the days of Ehrlich and Langley, when our understanding of receptor pharmacology began to emerge,1 the successful discovery of in vivo bioactive drug molecules has focused on two concepts: (i) identification and optimization of agents with good activity against a biological target(s) or pathway and (ii) finding a safe and well tolerated dose and regimen that maintain a high enough drug concentration at the intended site of action to elicit and sustain the desired pharmacological effect. Whether drugs have been developed through an intimate understanding of the specific protein targets being modulated or instead through a phenotypic approach, these two fundamental principles of favoring high equilibrium target occupancy and maintaining exposure in the diseased tissue have been central to guiding the discovery and optimization process. Note: For the purposes of the foregoing discussion, we will focus exclusively on design of inhibitory drugs as these are historically more common than agonistic agents. (Though perhaps counterintuitively, it is worth noting that Protac-induced protein degradation actually agonizes a process to achieve an inhibitory function.) Although focusing on the two principles of optimizing drug binding affinity and exposure has successfully provided a framework for the discovery of many highly effective medicines, this also brings implicit limitations. Identifying an agent with high potency and selectivity to modulate a biological target or process is not always straightforward, even for well described classes of proteins.2 This challenge may become more acute as greater genomic knowledge highlights new, less well characterized targets for drug intervention which often lack high affinity ligand binding sites. The second, but intimately linked, challenge of maintaining a sufficient drug concentration at the desired site of action for the required duration is also one that frequently causes drug discovery optimization teams much frustration, delay, ire (and expense) due to our limited ability to © 2017 American Chemical Society

Special Issue: Inducing Protein Degradation as a Therapeutic Strategy Received: August 27, 2017 Published: November 16, 2017 444

DOI: 10.1021/acs.jmedchem.7b01272 J. Med. Chem. 2018, 61, 444−452

Journal of Medicinal Chemistry

Perspective

to subvert this process and induce nonphysiological degradation. Nature has evolved highly efficient enzymatic systems to mediate the ubiquitinylation and subsequent degradation of proteins; if these could be harnessed and redirected for therapeutic benefit, the potential would be significant. However, in order to artificially mimic this process, a complex series of cellular events must be correctly orchestrated, the failure of any one of which will prevent the desired destruction of the target protein: (i) The agent designed to bring the target protein and ubiquitinylation machinery into close proximity must first access the appropriate intracellular compartment (e.g., cytoplasm or nucleus) in which both of the requisite protein partners reside. Given that the protein degrading agent needs to contain two independent binding moieties able to bind to the target protein and E3 ligase respectively, it is likely to be larger in size and cellular uptake may represent a challenge. (ii) Once inside the cell, the protein degrading molecule must then bind to the target protein and ubiquitinylation machinery simultaneously to form a ternary complex and enable ubiquitin transfer. The affinity of the degrading molecule to both proteins must allow a sufficient population of ternary complex to form in order to enable subsequent steps. The thermodynamics of ternary complex formation are less intuitively simple than binary receptor/ligand systems but have been well described.21 In particular, characteristic bell-shaped dose−responses (often termed a “hook effect”) leading to less efficient complex formation at high concentrations of the bridging molecule may lead to potential complications around in vivo dose selection. In some cases, secondary interactions directly between the two bound proteins can favor ternary complex formation through cooperativity or else disfavor it through undesired steric clashes.22 (iii) The ternary complex, once formed, must allow the two bound proteins to take up an appropriate conformation to allow suitable transfer of ubiquitin(s) residues to an appropriate acceptor site23 (frequently a surface lysine) with sufficient efficiency such that the rate of ubiquitin transfer is fast on the time scale of the intrinsic lifetime of the ternary complex. (iv) Induced ubiquitinylation of the desired substrate should also proceed at such a rate to overcome any competing removal of ubiquitins by deubiquitinase enzymes, of which there is a large family with varying substrate specificities.24 (v) The pattern of ubiquitin residues transferred to the substrate protein should then allow facile recognition by the proteasome to initiate the actual degradation. Proteins can be functionalized with multiple ubiquitins, forming chains of varying lengths and topologies linked through different lysine residues (e.g., K6, K11, K27, K29, K33, K48, K63, and M1). Physiologically, different polyubiquitin linkages and chain lengths/topologies can result in a range of cellular responses in a complex system of fine regulation of protein function. Of these, linear chains of K48-linked ubiquitin and others are thought to favor proteasomal recognition.25 The presence of a proteasome initiation region,26,27 a partially disordered loop or domain, is also thought to be important for

beyond the limitations of receptor pharmacology will offer great potential to medicine discovery. Induced protein knockdown using chimeric molecules designed to recruit the function of defined ubiquitin E3 ligases may represent one such access to novel small-molecule-induced pharmacology. This Perspective will highlight a number of key milestones as the concept has emerged and grown into one that could yet have far reaching impact on the drug discovery process. The discussion will focus specifically on degradation induced by proteolysis targeting chimeras (Protacs) and is necessarily far from comprehensive; the author humbly apologizes to all those researchers whose excellent work has not been included. For more in-depth literature surveys, the interested reader is referred to the multitude of excellent reviews that have appeared in recent months.5−11

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BORROWING FROM NATURE: INDUCTION OF BIOLOGICAL FUNCTION THROUGH PROXIMITY Nature has developed a plethora of pathways for maintaining biological homeostasis and cellular response to stimuli through careful potentiation and attenuation of signaling and other processes. Only a small proportion of the steps on these pathways however use direct small molecule modulators (e.g., hormones and other GPCR ligands, synaptic signaling, metabolite sensing and regulation); instead most regulation is carried by complex pathways ultimately resulting in modulation of protein synthesis or turnover. Since the 1990s, pharmacological intervention at the protein synthesis (DNA or RNA) level has been well described though continues to be challenging to translate into successful medicines.12 In contrast, aside from the notable clinical success of proteasome inhibitors13 that globally downregulate protein disposal routes, the removal of specific disease targets directly at the protein level has proved more challenging.14,15 As our understanding of the cell’s endogenous protein degradation mechanisms has however become clearer, potential therapeutic strategies have begun to emerge. Intracellular proteins are known to be degraded by two main routes, namely, the ubiquitin proteasome system (UPS) and autophagy/lysosomal routes. Through a series of pioneering studies from Ciechanover, Hershko, Rose, and others, ultimately culminating in the award of the 2004 Nobel Prize for Chemistry, the mechanisms of ubiquitin-dependent protein degradation have been steadily uncovered.16−18 The proteasome is a cellular complex responsible for the proteolytic degradation of proteins tagged for destruction via addition of specific chains of the 8.5 kDa protein ubiquitin. A full description of the dynamics of ubiquitin conjugation and signaling19,20 is beyond the scope of this article, but briefly, ubiquitin tags are delivered to target proteins via initial conjugation to an E1 ubiquitin-activating enzyme followed by transfer to a E2 ubiquitin-conjugating enzyme which then rely on a large family of adaptor proteins (ubiquitin E3 ligases) in order to deliver their ubiquitin cargo to the desired protein. Appropriately ubiquitin-tagged proteins are then recognized by the proteasome and proteolytically cleaved. Within this pathway, ultimate E3 ligase-mediated ubiquitin transfer to the target protein is driven by the proximity of the ligase complex in concert with specific recognition elements as well as the availability on the substrate of a suitable residue (often lysine) onto which the ubiquitin can be delivered. The rather promiscuous nature of this proximity-induced enzymatic ubiquitin transfer offers an opportunity to use artificial systems 445



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MetAP2 degradation in Xenopus extracts at high Protac concentrations. This landmark paper showed the feasibility of the concept but, due to the reliance on a highly charged peptide moiety for ligase recruitment, would be unlikely to give degradation in a cellular setting, and so the application to drug discovery was still far from clear. At the time, the work attracted modest interest from drug discovery groups despite the clear application to protein knockdown for chemical biology applications. Another interesting quirk of this early seminal paper resulted from the choice of an ovalicin derivative that bound to Met-AP2 through a covalent interaction. This reliance on a covalent linkage prevented one of the unique benefits of Protacs, namely, their catalytic effect (vide infra), potentially limiting the observed degradation efficacy. In the following years, a number of additional Protacs were characterized to degrade targets such as the estrogen receptor (ER) and androgen receptors (AR),34 though again the reliance on peptidic ligands required microinjection in order to demonstrate degradation within intact cells. The next important step in evolution of Protacs toward utility in drug discovery was when nonpeptidic E3 ligase binding moieties began to show evidence of degradation. In 2008, a nutlin, recently described to bind to the E3 ligase Mdm2, was combined with ligands to the androgen receptor to yield nonpeptidic Protacs able to give partial degradation of AR within cells at a concentration of 10 μM.35 Although the degradation was shown to be proteasomally dependent, from the limited data disclosed, it was difficult to assess if Mdm2 binding was required for degradation given that many AR ligands themselves spontaneously destabilize their cognate receptor or else allow it to be preferentially degraded.36 Although the field was clearly moving forward, application to drug discovery and medicinal chemistry was still limited mainly due to the modest level of cellular effects seen coupled with the perceived highly unusual chemical structures. These unfamiliar molecules were increasingly at odds with the contemporary focus on properties of drug molecules characterized most widely by such doctrine as the Lipinski “rule of 5”37 and a multitude of other medicinal chemistry property “guides” of varying levels of utility and relevance.38 More significant medicinal chemistry input and optimization would be required in order to determine if Protacs would remain a chemical biology tool or something more. A significant development in the further advancement of Protacs came in 2010−2012 with the identification of more drug-like E3 ligase binders39,40 by the groups of Craig Crews (Yale University and a long-standing leader in the field) and Alessio Ciulli (University of Cambridge, U.K., and an E3 ligase structural biology expert) and the subsequent initiation of a collaborative effort to also include a small group of drug discovery scientists at GSK (Stevenage, U.K.).41,42 Using knowledge gained from detailed X-ray and biophysical analysis of VHL bound to its substrate HIF-1α, fragment-based drug design principles were applied to successfully identify nonpeptidic ligands to mimic the natural peptidic VHL substrate.43,44 Between these groups, several series of binders to the E3 ligase von Hippel−Lindau (VHL) were identified, optimized, and incorporated into Protacs with the industry perspective now helping to drive the structure−activity relationships more quickly and resulting for the first time in Protacs showing highly potent cellular effects. The anatomy of an early Protac is exemplified in Figure 1 along with a number

proteasomal processing, and in some cases where this region is absent, proteins may be extensively polyubiquitinated but still not recognized by the proteasome.28 (vi) If all of the above steps have been satisfied and the desired protein degraded within the proteasome, this still does not ensure that steady state levels of protein will be diminished. The induced degradation rate must be significantly faster than the de novo resynthesis rate of the protein, a variable that may differ widely between proteins and cell types.29,30 Similarly, if feedback mechanisms triggered by the loss of mature protein upregulate transcription or translation of new protein, initial lowering of equilibrium protein levels may be lost over time. Overall, any drug intervention to subvert the endogenous protein regulation machinery to remove a specific protein requires a high degree of design and specificity to mediate a number of biological steps, a significant challenge. The journey to define, understand, and overcome this series of challenges and to achieve successful protein degradation with drug-like molecules has taken over 20 years, and a number of notable advances in the field are now briefly summarized.

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FROM CONCEPT TO PRACTICE One of the earliest reports of artificially induced protein degradation came in 1995 with the design of a series of modified E2 conjugating enzymes (such as TaUBC4) fused to Ig-binding motifs. The result was a series of ∼250 amino acid fusion proteins able to induce transfer of ubiquitin directly from the E2 to specific substrates.31 Furthermore, the resulting ubiquitinylated substrates were shown to undergo modest proteasomal processing in a cell lysate showing for the first time that these artificially induced ubiquitinylation signatures could be recognized by the proteasome resulting in induced degradation. This landmark observation proved that substrate ubiquitinylation could be induced by artificial adaptors, but the direct therapeutic application of these large and relatively inefficient fusion proteins was limited. A further conceptual contribution came from the 1999 patent application from Proteinix32 which claimed a series of bifunctional inducers of protein degradation which now relied on much smaller “ubiquitinylation recognition elements” giving agents now with molecular sizes down to around 1 kDa. No specific data were disclosed, so it is difficult to assess the efficiency of these molecules and indeed if they actually gave any meaningful degradation as claimed, but they do represent the first claimed conceptual examples of non-protein-based agents with potential to induce protein degradation. The lack of specific disclosed data and the fact that this work was not subsequently described in the primary literature perhaps explain why it had little impact on the field at the time. A more significant and well described advance came in 2001 with the disclosure from the Deshaies and Crews laboratories33 of cellular degradation of the aminopeptidase MetAP2 using a hybrid of the small molecule MetAP2 inhibitor ovalicin linked to a IκBα phosphopeptide epitope known to bind to the ubiquitin E3 ligase SCFβTrCP. This disclosure, which introduced for the first time the term proteolysis-targeting chimera, or Protac, elegantly demonstrated the formation of a ternary complex between the degradation target, MetAP2, and SCFβTrCP, resulting in rapid (30 min), proteasomally dependent 446



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Figure 1. An exemplar Protac45 showing the three basic units of target-binding ligand, linker, and E3 ligase ligand. Initially, these unusual chemical structures attracted a wide range of questions from the drug discovery community. Many of these questions are now beginning to be answered in the affirmative.

Figure 2. Schematic of Protac action. Protacs bring the target protein and ubiquitinylation machinery into close proximity and catalytically mediate initiation of the degradation cascade.

of the questions posed as initial Protac data began to emerge. As a direct result of the advances made in this industry− academia collaboration, the Yale spinout company Arvinas, which has now established itself as a significant innovator in the field, was founded in 2013. As members of the Crews group also took their expertise to other academic centers, the early success of Protac work was set to grow rapidly. Unlike earlier VHL ligands, these moieties developed by the GSK/Yale/Cambridge groups were nonpeptidic (reducing potential protease-mediated instability), smaller, and not excessively polar and thus offered the potential for incorporation into Protacs with more drug-like properties. Indeed, when linked to a ligand to receptor-interacting protein kinase 2 (RIPK2) in 2013, a Protac was discovered that for the first time gave highly potent cellular effects now around 1 nM,45 several orders of magnitude more active than previous molecules. Importantly, the E3 ligase dependence of the degradation was confirmed using a VHL-nonbinding enantiomeric control which was unable to recruit the ligase function and, as expected, gave no degradation. The mechanism of action

(Figure 2) was further confirmed by demonstration of formation of the postulated ternary complex, and through elegant use of radiolabeled substrate, the action of the Protac was proven for the first time to be catalytic, with a single Protac molecule shown to mediate the degradation of multiple RIPK2 substrate molecules. This key result confirmed a unique facet of Protac action which differentiates the approach from most other small molecule pharmacological interventions, namely, “event-driven” rather than occupancy-driven pharmacology. The mechanism was also shown to be highly selective. Using expression proteomics to quantify around 7000 cellular proteins, a very high level of selectivity for degradation of the desired RIPK2 over other proteins was observed. These data had suddenly elevated Protacs into a technology that now had the potential to deliver highly effective pharmacology in cells, in vivo and even in patients. Almost simultaneously to the GSK-Crews paper, independent publications from the Bradner,46 Arvinas,47 and Ciulli48 groups showed degradation of the BET family of epigenetic bromodomain-containing proteins using the ligases cereblon 447



Perspective

typically associated with efficient membrane crossing,52,53 Protacs attain sufficient intracellular concentrations which, in concert with the catalytic mechanism, drive nM to pM levels of cellular potency. The larger size of Protacs relative to traditional small molecule agents may result in a slower rate of membrane crossing, but the ultimate concentration of drug able to access the cell is clearly adequate to drive highly potent effects. The exact mechanism of cellular uptake has not been definitively characterized, but the observation that a very wide range of chemotypes of Protacs are able to penetrate a wide range of different cells is suggestive of a passive process not reliant on any specific active routes of uptake. 2. Selectivity. A key goal of much contemporary medicinal chemistry is the design of highly selective molecules, minimizing binding to off-target proteins which may lead to unwanted pharmacology and toxicity. Strategies including structure-based design and parallel assay data generation now routinely allow impressive levels of selectivity to be achieved, even in cases of families of targets with highly homologous binding sites (e.g., kinases). Despite these advances, achieving good selectivity over specific off-targets is frequently still a significant challenge. Although the in vivo selectivity of pharmacological agents is largely determined by the ratio of binding Kd values to the bioactive forms of the desired target and other off-targets, other factors including differential tissue distribution and competition with endogenous cofactors with differing Km values also contribute to effective selectivity. Additional opportunities for gaining functional selectivity can be seen with modulators of certain GPCRs, ion channels, and nuclear hormone receptors where ligands can be designed to demonstrate agonism/ antagonism (or inverse agonism) or opening/closing effects on different targets, thus introducing selectivity of function even when selectivity of binding between protein targets is not possible.54 Targeted protein degradation offers an additional strategy to modulate the functional selectivity of a ligand. In the case of incorporating a highly selective ligand into a Protac, highly selective degradation is often observed as seen with Protacs targeting RIPK245 and the BET family.46 However, the intrinsic binding selectivity of a ligand may also be overcome using appropriately designed Protacs which can result in more selective degradation. Preliminary reports showed Protacs with modest claimed preferential degradation of BRD4 over other BET family members despite using JQ1, a ligand with no intrinsic binding preference.48 More recent work however has used X-ray crystallographic analyses to suggest the Protac linker may yield productive secondary interactions leading to selective stabilization of the BRD4-containing ternary complex and subsequent selective degradation.22 Related effects in induced selectivity have also been noted in other areas including kinase degradation.55 If these interactions can be prospectively designed, further, hitherto inaccessible levels of functional selectivity may become achievable. As well as selective stabilization of specific ternary complexes, additional Protac-induced selectivity may also be achieved through accessing specific ternary complex geometries that favor ubiquitin transfer to some substrates over others or by taking advantage of the likelihood that protein targets in different subcellular localizations will have differing abilities to be trafficked to and degraded by the proteasome. 3. Broad Applicability across Cells and in Vivo Systems. Ubiquitin ligases are expressed widely across cell types; the ubiquitin proteasome system is largely conserved

and VHL, respectively. In particular, the incorporation of binders to the E3 ligase cereblon into Protacs represents a valuable new usage for molecules known since the 1960s49 whose mechanism of action has been exquisitely delineated in more recent years.50,51 These four papers45−48 published in quick succession in the summer of 2015 announced the arrival of Protacs as, for the first time, an approach with real application to drug discovery. At a stroke, the level of investment and interest in Protac approaches increased significantly and attention began to turn to the multitude of questions that would now need to be addressed in order for Protac ideas to be tested in the unforgiving environment of active drug discovery and development groups.

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REALIZING THE ADVANTAGES OF DEGRADATION OVER INHIBITION Protacs offer a range of unique potential advantages relative to traditional small molecule antagonists that include (i) high cellular potency driven by the catalytic mode of action, (ii) highly selective degradation, (iii) wide applicability across cells and in vivo systems, (iv) potential for extended pharmacodynamic duration of action, and (v) the opportunity to mediate novel pharmacology. Progress has now begun to show the practical realization of these opportunties. 1. Cellular Potency and Catalytic Effect. As noted above, most small molecule-induced pharmacology is driven by the requirement for maintenance of high equilibrium target occupancy which in turn often requires high drug dose. Such high dose agents have historically been associated with a higher likelihood of exhibiting (often off-target) toxic effects, greatly slowing and complicating the drug discovery process. Protacs, acting as agonists to catalytically initiate a degradation cascade, are no longer limited by this paradigm. Instead, a low level of target protein occupancy may be sufficient to maintain a rate of protein degradation that quickly depletes a high proportion of cellular protein giving the desired pharmacological effect. This catalytic mode of action means that frequently, cellular EC50 values much lower than the intrinsic target-binding Kd can be observed.45,47 This is in contrast to traditional antagonism where a significant loss in potency can be seen on going from in vitro binding Kd to cellular potency. A further consequence of the catalytic nature of Protac action is an often profound timedependence of degradation with greater protein depletion seen over time. The catalytic effect also offers the additional advantage that desired protein degradation may be observed at Protac concentrations much lower than those required to give high levels of E3 ligase inhibition. Protac_RIPK245 binds to its cognate ligase VHL with Kd ≈ 0.5 μM while delivering degradation of RIPK2 with DC50 ≈ 1 nM (DC50 is the concentration at which 50% of cellular protein is depleted). Full inhibition of VHL function may lead to undesirable effects on, for example, regulation of hypoxic sensing, but concentrations of Protac_RIPK2 of >10 μM are required to achieve sufficient VHL inhibition to disrupt its pharmacology (as judged by HIF1α stabilization) thus leading to a significant window between desired degradation-induced pharmacology and unwanted inhibition-induced effects.45 Historically, the design of higher molecular weight (MW > 1000 Da) compounds that penetrate cells with high efficiency has been perceived as challenging. Despite molecular weight and polar surface area values well beyond those properties 448



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chronic conditions, can be extremely poor,61 severely limiting the therapeutic benefit. Protacs, which can be delivered by multiple routes, may offer many attractive clinical dosing options. 5. The Opportunity to Mediate Novel Pharmacology. Many drug targets present particular challenges to drug discovery. They may not have a specific catalytic active site whose function can be blocked, instead relying on larger protein interfaces to mediate signaling. Alternatively, proteins may possess multiple functions and catalytic domains, blocking of only one of which may have partial or no efficacy. Although identifying high affinity inhibitors may be a difficult or impossible strategy for these targets, a Protac-induced degradation may offer a solution, as binders (in contrast to functional inhibitors) only are needed to facilitate E3 ligase recruitment and initiation of the degradation cascade. As the ligand itself need not possess a specific function, it can bind anywhere on the target protein (or indeed on another protein in complex with the target), greatly increasing the probability of finding a suitable ligand when using appropriate binding site agnostic (biophysical) screening approaches. Alternatively, ligands to a druggable site distal to a desired functional domain may be used as an anchor to facilitate degradation of the complete protein including any unligandable sites. A further advantage that Protacs offer over corresponding inhibitors to a specific target is their ability to phenocopy genetic knockdown, an approach increasingly used to implicate specific drug targets in disease etiology. This represents a unique opportunity where Protacs can induce more profound pharmacology which will present additional options for increased therapeutic benefit and potentially greater phase II success rates. This ability of Protacs to offer potentiated pharmacology should be used with care of course. Pharmacology that is beneficial is considered “efficacy”, while all other pharmacology may similarly be considered as “toxicity”. Increasing both of these in parallel may offer little benefit, though the selective potentiation of one over the other offers tremendous potential. Removal of the entire protein instead of inhibition of a single function can also lead to significantly improved pharmacology, as noted with BET degraders which show much more profound antiproliferative effects than the inhibitory ligands on which they are based.56 Although this potentiation or introduction of additional efficacy may offer greater therapeutic effect, it may also introduce unwanted effects if essential cellular functions are also modulated by protein removal. Protac-induced potentiated pharmacology should be used with care.

across mammalian species. As a result, Protac-induced degradation has been observed across a wide range of both human and rodent cells and in healthy and diseased tissues suggestive of broad applicability. Translating Protac cellular effects into in vivo and ultimately clinical systems is a central challenge, and the in vivo efficacy of Protacs is likely to rely on complex pharmacokinetic/pharmacodynamic relationships dependent on a number of factors: The intrinsic pharmacokinetic profile needs to be suitable to allow sufficient drug exposure for long enough to achieve protein knockdown. So far, limited Protac in vivo data are available, although there are indications that Protacs can have good metabolic stability and tissue distribution.56−58 As more data emerge, it will be interesting to see whether Protacs will share the typical oxidative routes of metabolism seen with their constituent ligands or if different metabolic routes or clearance mechanisms are introduced. The larger molecular size of Protacs may alter their ability to access hepatocytes or other sites of metabolism or to bind in CYP450 active sites. Contrarily, the presence of the linker may present additional sites for potential metabolism requiring careful design to achieve acceptable metabolic stability. 4. Potential for Extended Pharmacodynamic Duration of Action. The degradation of target proteins is time dependent and can be fast, with Protacs depleting cellular protein to close to basal levels within minutes in favorable cases.45,59 Once the pre-existing reservoir of cellular protein is depleted, however, the Protac need only degrade the smaller pool of de novo resynthesized protein to maintain knockdown; for many proteins with modest turnover rates, protein is resynthesized only slowly29 potentially allowing modest tissue concentrations of Protac to maintain efficient knockdown. Even after complete Protac clearance, cells may still take a significant period of time to recover the pool of protein to a level sufficient to re-establish physiological signaling resulting in a greatly extended duration of action. In favorable cases, a Protac that can rapidly deplete the entire pool of a slowly resynthesized protein could deliver significant pharmacology from short-term drug exposure with a subsequent duration of effect of the order of hours/days delivering sustained efficacy despite the drug being present at low or even undetectable levels for a significant proportion of the dosing interval. This profile, coupled with the catalytic effect allowing high potency, has the potential to allow excellent knockdown-induced pharmacology driven by low drug exposures (Cmax and AUC), low dose, and long dosing interval. If indeed Protac dosing regimens can allow infrequent administration, a range of dosing routes well suited to the pharmacodynamic profile may be used. In common with biological agents, dosing via the subcutaneous or intramuscular route may be attractive where pharmacological effect can be maintained for 1 week or longer. Many other formulations are increasingly being used for controlled release, and these too may be well suited for Protac action. Although intravenous administration also remains an acceptable route for appropriate indications, increasingly there is growing interest and progress in the identification of orally available Protac agents. Despite their large molecular size, some Protacs remain close to meeting the well-known Lipinski rules and, with additional use of prodrug strategies60 or other formulations, may offer significant potential for oral delivery. Although oral delivery is often considered the preferred route of delivery for small molecule medications, patient compliance, especially for more

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WHERE NEXT FOR DRUG DISCOVERY? Protac-induced protein degradation has yielded impressive preliminary efficacy in a limited number of cellular and in vivo systems, but its broader utility and application in a clinical setting is yet to be tested. What is the likelihood that Protacmediated protein degradation will be widely applicable across protein families? Other, well described examples of small molecules that potently mediate the degradation of their cognate targets such as the androgen receptor62 and estrogen receptor63 rely on target-specific mechanisms of action unlikely to be readily applied to other targets. In contrast, examples of successful Protac-mediated degradation so far have been applied to diverse and unrelated targets. From proteases to nuclear hormone receptors, epigenetic factors, and kinases, the scope of this approach has the potential to extend to a significant proportion of the estimated 80% of the proteome 449



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which can be regulated by proteasomal processing.64 Exactly which protein substrates are most amenable to the approach, which others may be refractory, and what are the factors that determine this will be the topic of much active research in coming years. Large scale proteomic analyses may allow much more significant data sets to be assembled in order to shed further light on this question. The flexibility of the approach is also being steadily expanded by the use of new ubiquitin E3 ligases. While most nonpeptidic Protacs have used the E3 ligases VHL and cereblon, more recent reports have shown greater use of members of the IAP family of ligases.58,65 Mdm2 may also be a suitable ligase but has so far attracted fewer disclosures. Across these successfully utilized ligases, a range of different ligands are available for incorporation into Protacs offering broad opportunities to prepare different chemical series, each with tunable properties and abilities to produce ternary complexes with differing geometries. Although the current small set of effective ligases have shown tremendous utility in demonstrating feasibility of Protac knockdown, each ligase brings with it potential issues including the risk of undesired ligase-mediated pharmacology or intrinsic limitations (eg metabolic instability) of the ligasebinding ligands themselves which must be addressed to enable clinical evaluation. As a result, attention will likely also turn to identifying ligands to additional E3 ligases for inclusion in next generation Protacs. There are estimated to be around 600 E3 ligases,66 though so far, only a tiny proportion of these have proved amenable to small molecule ligand discovery. Future efforts to identify ligands for additional ligases with different tissue distributions or endogenous substrate specificities may yet yield additional options for specific intervention. Direct recruitment of E2 conjugating enzymes or even the proteasome itself67 may also represent alternative strategies to create future Protac-like molecules. A critical additional aspect in designing more effective Protacs lies in the linker which not only disposes the targetbinding and E3 ligase-binding termini appropriately (influencing degradation efficiency as discussed above22) but also has a significant influence on the overall molecular properties of the Protac, a factor that is paramount in achieving appropriate biological function via the right combination of cellular uptake, ternary complex geometry, stability, and aqueous solubility. Indeed, appropriate choice of linker and linking points remains one of the most important aspects of Protac design, though, as perhaps this represents some of the most valuable know-how in the field, extensive structure−activity relationships in this domain have been slow to appear in the literature. Between the choices of E3 ligase ligand, the target-binding ligand, and both the identity and attachment positions of the linker, there are a number of opportunities to design both very good, and very bad, Protacs in much the same way as with traditional small molecule medicinal chemistry. The concept of an “allpurpose” combination of a binder to a specific E3 ligase and linker which can be appended to any ligand to ensure optimal degradation is unlikely to become reality. Further challenges yet lie ahead to successfully translate the promise of Protacs into a clinical setting including chemical synthesis on a multikilogram scale and CMC (chemistry, manufacturing, and controls) considerations. Many Protac molecules possess a combination of characteristics, such as the requirement for a long synthetic sequence, presence of multiple stereocenters, challenging analytical characterization (due to complex solution confirmations), and potential for non-

crystallinity each of which has the potential to complicate drug development. Although drug development has been adept at tackling each of these problems in isolation, the combination of these factors may require novel strategies to streamline development. The modular nature of Protac architecture may offer potential for highly convergent routes,68 and if low dose can indeed be achieved, some of these production issues may be attenuated. Given the conformational flexibility likely in most Protacs, it is also unclear if they will form stable crystalline forms, potentially adding to the emerging number of examples of amorphous drugs now being developed.69 Reports of clinical studies using Protacs have yet to emerge, and it is only when data from such studies become available that we will truly begin to see the therapeutic potential of these chimeric catalysts. Many new approaches to drug discovery have promised to transform the medicine landscape only to be thwarted when faced with extensive preclinical and clinical safety testing required even before a phase II evaluation of efficacy is possible. Time will tell if Protacs suffer a similar fate or whether they can plot a path through this complex phase of the drug discovery process. If the high cellular potency often seen can be translated to low clinical doses, then good protein knockdown at well tolerated doses may be possible giving, with well-chosen targets, real benefit to patients. The Protac journey from concept to potential clinical evaluation has stretched over 20 years but has accelerated greatly in recent years with groundbreaking work from academic leaders and forward-looking pharma and biotech groups alike. The underpinning science offers a real opportunity to break free of the shackles of receptor pharmacology which has limited much of modern small molecule drug discovery. The field is now poised to answer some of its most critical questions to see if these novel scientific concepts can indeed translate to agents that deliver real clinical benefit and unprecedented medicine opportunities. We wait expectantly.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +44 7785 720453. E-mail: ian.churcher@benevolent. ai. ORCID

Ian Churcher: 0000-0001-6903-5824 Notes

The author declares no competing financial interest. Biography Ian Churcher is currently VP of Drug Discovery at BenevolentAI where he is applying a range of artificial intelligence approaches to all phases of drug discovery and development. Prior to this, for 5 years, he was Head of the Protein Degradation Discovery Performance Unit at GSK, one of the pioneering groups that, working with Prof Craig Crews, demonstrated the application of Protac approaches to drug discovery. Ian has previously held a number of drug discovery and medicinal chemistry roles in GSK and Merck where he has always championed novel technologies and strategies to discover drugs. Ian was also a Visiting Professor in the Department of Chemistry at the University of Oxford from where he also holds M.A. and D.Phil. degrees.

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ACKNOWLEDGMENTS I thank Ian Smith for assistance in preparing figures, Chris Tinworth for assistance in preparation of the manuscript, and 450



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Mark Rackham for his thoughtful comments. I also thank all the dedicated scientists who have contributed to allowing the protein degradation field to move so quickly both within my own group and across industry/academia.

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ABBREVIATIONS USED

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REFERENCES

AR, androgen receptor; AUC, area under the curve; BET, bromo- and extra-terminal domain; CMC, chemistry, manufacturing, and controls; ER, estrogen receptor; IAP, inhibitor of apoptosis protein; Protac, proteolysis-targeting chimera; RIPK2, receptor-interacting protein kinase 2; UPS, ubiquitin proteasome system; VHL, von Hippel−Lindau

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Protac-Induced Protein Degradation in Drug Discovery: Breaking the

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