Proteolysis targeting chimeras (PROTACs) in ‘beyond rule-of-five’ chemical space: Recent progress and future challenges

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Digest

Proteolysis targeting chimeras (PROTACs) in ‘beyond rule-of-five’ chemical space: Recent progress and future challenges

T

Scott D. Edmondsona, , Bin Yanga, Charlene Fallanb ⁎

a b

Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Boston, MA, USA Medicinal Chemistry, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK

ARTICLE INFO

ABSTRACT

Keywords: PROTACs Degradation Beyond rule-of-five bRo5 Proteolysis targeting chimeras Absorption Oral bioavailability

Proteolysis targeting chimeras (PROTACs) are heterobifunctional compounds with molecular weights and other properties that lie outside the classic ‘rule-of-five’ space. Consequently, PROTACs have unique challenges associated with their development as potential therapeutic agents. This review summarizes and analyzes a representative set of recent PROTACs and highlights some of the potential future challenges facing this promising modality.

Introduction The development of new therapeutic modalities beyond small molecule drugs has made significant progress in recent decades. Advances have largely been driven by the use of monoclonal antibodies1 and to a lesser extent, in antisense oligonucleosides (ASOs)2 and small interfering RNA (siRNA).3 Other rapidly advancing modalities, such as cyclic or stapled peptides,4 aptamers,5 modified messenger RNA (mRNA),6 or CRISPR-Cas9,7 face challenges including cellular permeability, metabolic stability, therapeutic index and/or difficulties with oral drug delivery. Limitations are also evident with traditional small molecules as protein function inhibitors, including off-target pharmacology (that can limit dosing regimens) and the absence of potent small molecule ligands for the majority of cellular targets in the proteome.8 Nevertheless, several advantages of small molecules set them apart from newer therapeutic modalities, including: 1) intrinsic cellular permeability for engagement of intracellular targets; 2) oral bioavailability for treatment convenience and/or 3) lower manufacturing costs.

Proteolysis targeting chimeras (PROTACs) are an emerging modality with the promise to overcome some of the shortcomings of small molecules while retaining their advantages to directly engage intracellular targets, potential for oral bioavailability and reduced manufacturing challenges compared to antibodies or oligonucleotides. As heterobifunctional molecules, PROTACs consist of three key structural components; a protein-of-interest (POI) ligand, a ubiquitin E3 ligase ligand, and a linker to join these together. Instead of relying on target occupancy to disrupt specific protein functions, PROTACs catalyze protein degradation by recruiting ubiquitin E3 ligases to promote polyubiquitination of the POI and its subsequent degradation in the proteosome through the ubiquitin-proteosome-system (UPS, Fig. 1). This mechanism of action harbors intriguing potential for disease intervention beyond that which may be achieved with small molecules.9 For example, a reduction in protein concentrations in disease tissues may result in benefits beyond simple inhibition of protein function. Furthermore, PROTAC-mediated degradation requires only a POI binder and not a functional inhibitor; therefore, PROTACs could have

Abbreviations: PROTAC, proteolysis targeting chimeras; ABL, Abelson murine leukemia viral oncogene homolog 1; ADME, absorption, distribution, metabolism, and excretion; ALK, anaplastic lymphoma kinase; AR, androgen receptor; BCL6, B-cell lymphoma 6 protein; BET, bromodomain and extra terminal domain; BRD2, bromodomain-containing protein 2; BRD3, bromodomain-containing protein 3; BRD4, bromodomain-containing protein 4; BRD7/9, bromodomain-containing proteins 7/9; BTK, Bruton’s tyrosine kinase; CDK8, cyclin-dependent kinase 8; CDK9, cyclin-dependent kinase 9; CRBN, cereblon; EPSA, exposed polar surface area; ERRα, estrogen-related receptor alpha; ERα, estrogen receptor α; FAK, focal adhesion kinase; FKBP12, the 12-kDa FK506-binding protein; FLT3, Fms like tyrosine kinase 3; HDAC6, histone deacetylase 6; IAP, inhibitor of apoptosis proteins; MDM2, mouse double minute-2; PBS, phosphate-buffered saline; PCAF, P300/CBPassociated factor; PI3K, phosphoinositide 3-kinase; PK/PD, pharmacokinetic/pharmacodynamic; POI, protein-of-interest; RIPK2, receptor-interacting serine/threonine-protein kinase 2; Ro5, Lipinski rule-of-five; SC, subcutaneous injection; IP, intraperitoneal injection; SNIPERs, specific and nongenetic IAP-dependent protein erasers; TBK1, TANK-binding kinase 1; UPS, ubiquitin-proteosome-system; VHL, von Hippel–Lindau tumor suppressor ⁎ Corresponding author. E-mail address: [email protected] (S.D. Edmondson). https://doi.org/10.1016/j.bmcl.2019.04.030 Received 13 February 2019; Received in revised form 11 April 2019; Accepted 16 April 2019 Available online 20 April 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

interactions supported a structure guided approach to identify AT1 (10)29 which also degrades BET proteins but with improved selectivity for BRD4 degradation compared to earlier PROTACs. A change in the bromodomain POI ligand to incorporate a different exit vector was also shown to tune the degradation of the BET PROTACs (MZP54, 11) to be BRD3/4 selective.30 Outside the BET family, VHL-PROTACs have been shown to degrade a variety of additional targets including ERRα (12),31 RIPK2 (13),31 ABL (14, DAS-VHL),32 FLT3 (15),33 FAK (16),34 and TBK1 (17).35 Linkers are typically variations of ethers (e.g. PEGs) and/ or hydrocarbon chains attached to the POI ligands through an amide, ether, or amine. The specific attachment point from the VHL ligand is similar for the majority of these – through an amide bond to the (S)-tertleucine moiety. More recently, however, new vectors have been reported from this hydroxyproline VHL ligand for VHL-PROTAC that degrade BRD7/9 (18, VZ185)36 and AR (19, ARD-69).37 Early examples of PROTACs bearing a cereblon (CRBN) E3 ligase warhead were dBET1 (20)38 and ARV-825 (21)39 which both selectively degrade BRD4. These CRBN-PROTACs leverage thalidomide and pomalidomide derivatives as the E3 ligand, respectively. In matched pair comparisons of lenalidomide vs pomalidomide, improved in vitro degradation was reported with lenalidomide as the E3 binder for the structurally distinct BET PROTACs BETd-260 (22)40 and QCA-570 (23).41 Similar to VHL-PROTACs, the CRBN-PROTACs can also degrade a wide variety of substrates such as BCL6 (24),42 CDK8 (25),43 CDK9 (26, 27),44,45 PI3K (28),46 BTK (29–31),47,48,49 ALK (32),50 HDAC6 (33),51 sirtuin (34),52 PCAF/GCN5 (35),53 pirin (36),54 and FKBP12F36V (37).55 Recently, Zhang and co-workers have demonstrated that potent Rapamycin based PROTAC, RC32 (38),56 facilitates protein degradation in mice via oral administration (60 mg/Kg, BID) whereas IP dosing was used for rats, pigs and rhesus monkeys. As in the case with VHL-PROTACs, linkers are mostly comprised of alkyl or alkoxy groups and often contain an additional functional group to attach the POI ligand to the E3 warhead such as amides or triazoles. One notable exception is compound 23, with acetylenes on either side of the pyrazole moiety in the linker.

Fig. 1. PROTAC mode-of-action and anatomy.

the ability to degrade proteins previously believed to be ‘undruggable’ through conventional small molecule inhibition. Oral dosing is the preferred route of delivery of therapeutic agents in most chronic disease settings. A formidable challenge preventing PROTACs from realizing their therapeutic potential is their lack of compliance with the well-established drug-like properties associated with oral drugs, namely Lipinski’s “rule-of-5” (Ro5). A number of reviews have commented on recent progress in PROTACs,10–15 and in this digest, we focus on oral bioavailability of the molecules from a medicinal chemistry perspective. Oral bioavailability is a function of the fraction of the drug escaping gut and hepatic metabolism as well as the fraction absorbed, and PROTACs face challenges with each of these parameters. Additionally, potential broader ADME challenges to transform PROTACs from preclinical tool compounds to medicines will be discussed. Breadth of chemical matter A wide variety of target classes have now been successfully degraded through PROTACs (Table 1), with a significant number of reports in the past 2 years.10,16,17 To date, the majority of degraders rely on the engagement of a small group of E3 ligases with known ligands. One of the earliest small molecule PROTACs leveraged the mouse double minute-2 (MDM2) E3 ligase and its binding ligand (nutlin derivative) to degrade the androgen receptor (AR) (1).18 Although this provided proof-of-concept for a bifunctional molecule to hijack the UPS to degrade a POI, 1 was not a very potent degrader. Recent work to optimize the MDM2 ligand was reported using an idasanutlin derivative as the MDM2 binder and this resulted in identification of a potent BRD4 degrader (2).19 It is worth mention that these MDM2 ligands are not trivial to access synthetically and this may play a role in their limited use to date. Ligands that bind to the inhibitor-of-apoptosis (IAP) E3 ligase have also been reported to induce potent degradation. Early IAP-PROTACs used a bestatin ligand, but these compounds were not very potent.20 More recently, a series of PROTACs that use a derivative of LCL-161 were reported to degrade multiple targets, including BRD4 (3), ABL (4), AR, and the estrogen receptor α (ERα, 5).21,22 This sub-series of PROTACs has been dubbed SNIPERs (specific and nongenetic IAP-dependent protein erasers). Further optimization on the E3 binding moiety afforded ERα degrader 6 (A1874), which was more potent than 5 both in vitro and in vivo.23 Peptidic ligands to the Von Hippel-Lindau (VHL) tumor suppressor E3 ligase have been known for some time,24 but small molecule approaches to hijack VHL with PROTACs was demonstrated only recently using a hydroxy proline derived binding site ligand.25 Early success was demonstrated through selective degradation of BRD4, one of the BET bromodomain family transcription factors. MZ1 (7) is an early example that demonstrated selective BRD4 degradation despite the use of a BRD4 ligand that is non-selective over BRD2 or BRD3.26 Compound 8 is structurally similar to 7 but with a triazole linker that can be conveniently assembled using click chemistry.27 Linker optimization and a small modification at the VHL binding moiety produced a potent in vivo probe compound 9 (ARV-771) which was efficacious at degrading BRD4 in rodents.28 An X-ray crystal structure of some key VHL binding

Protacs and oral absorption challenge In recent years, a number of drugs have progressed that lie in chemical space beyond that described by Lipinski’s Ro557 and/or Veber’s rotatable bond/polar surface area58 guidelines. These ‘beyond rule-of-5’ (bRo5) drugs often possess molecular weights above 700 and/or other violations of Lipinski/Veber’s rules. Despite these rule violations, many of these compounds exhibit adequate oral bioavailability for further development as oral drugs. When gut and hepatic metabolism are low, oral bioavailability is primarily a function of the absorbed drug fraction, and the Ro5/Veber guidelines were intended to help provide descriptors predictive of drug absorption. In recent years, several groups have analyzed different bRo5 compound sets to better define in silico descriptors of absorption for compounds in this unique chemical space.58,59 For example, Doak, et al. have collated some of the upper boundary limits of key descriptors originally described by Lipinski and Veber,60 and several groups have recently demonstrated that compounds which lie in this space can exhibit oral absorption and have attempted to relate these to various composite in silico properties. One such composite score described by Degoey, et al is the AB-MPS metric which is calculated using cLogD, the number of aromatic rings (nAr), and the number of rotatable bonds (nRotB) according to the formula AB-MPS = Abs(cLogD −3) + nAr + nRotB.61 The lower the AB-MPS score, the more likely the compound is to be absorbed, and a value of ≤14 is reported to predict a higher probability of oral absorption. Kihlberg, et al. have analyzed the upper boundary parameters from several groups and plotted them in a radar plot compared to the original Veber/Ro5 parameters.62 Their analysis demonstrates that these upper boundaries can vary somewhat depending on the molecules contained within the compound sets analyzed, though some parameters 1556

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

Table 1 Representative recent PROTACs organized by E3 warhead: MDM2, IAP, VHL, and CRBN binders. PROTAC

Structure

Name

Target (POI)

E3 Ligase

DC50/Dmax

Cellular/in vivo

Ref

1

Compound 14

AR

MDM2

10 μM/–

In vitro

18

2

A1874

BRD4

MDM2

32 nM/98% at 100 nM

In vitro

19

3

SNIPER(BRD4)-1

BRD4

IAP

> 3 nM & < 10 nM/70% at 10 nM

In vitro probe

20

4

SNIPER(ABL)-39

ABL

IAP

> 3 nM & < 10 nM/ > 90% at 100 nM

In vitro probe

21

5

SNIPER(ER)-87

ERα

IAP

> 1nM & < 3 nM/70% at 10 nM

In vivo efficacy (IP injection)

22

6

SNIPER(ER)-110

ERα

IAP

< 3 nM/80% at 100 nM

In vivo probe

23

7

MZ1

BRD4

VHL

< 100 nM (BRD4)/ > 96% at 50 nM

In vitro cellular probe

26

8

12b

BRD4

VHL

0.083 μM/–

In vitro

27

9

ARV-771

BRD4

VHL

< 5 nM for BRD2/3/4/ > 99%

In vivo (SC) efficacy

28

10

AT1

BET

VHL

> 10 nM & < 100 nM for BRD4 short/ > 90%

In vitro prob

29

11

MZP54

BET

VHL

10 nM- < 100 nM/87% at 50 nM

In vitro cellular prob

30

12

PROTAC_ERRα (1)

ERRα

VHL

100 nM/86% at 1 μM

In vivo probe (IP injection)

31

(continued on next page)

1557

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

Table 1 (continued) PROTAC

Structure

Name

Target (POI)

E3 Ligase

DC50/Dmax

Cellular/in vivo

Ref

13

PROTAC_ RIPK2

RIPK2

VHL

1.4 nM/ > 95% of 10 nM

In vitro

31

14

DAS-VHL

ABL

VHL

1 μM/ > 65% at 1 μM

In vitro

32

15

FLT-3 PROTAC

FLT-3

VHL

< 5 nM/ > 90% at 50 nM

In vivo PD probe (IP injection)

33

16

PROTAC-3

FAK

VHL

3 nM/ > 99% at 50 nM

In vitro cellular prob

34

17

Compound 3i

TBK1

VHL

12 nM/96%

In vitro cellular prob

35

18

VZ185

BRD7/9

VHL

< 5 nM (BRD9)/95%

In vitro cellular probe

36

19

ARD-69

AR

VHL

< 1 nm/ > 95% at 10 nM

In vivo probe (IP injection)

37

20

dBET1

BRD4

CRBN

< 100 nM (BRD4)/95% at 1 μM

In vivo probe (IP injection)

38

21

ARV-825

BRD4

CRBN

< 1 nM for BRD4/ > 99% at 1 nM

In vitro probe (IP injection)

39

22

BETd-260

BET

CRBN

< 1 nM/ > 95% at 1 nM

In vivo PD/ efficacy probe (IV dose)

40

23

QCA-570

BET

CRBN

< 1 nM BRD2/3/4/ > 99% at 0.5 nM

In vivo PD /efficacy probe (IV dose)

41

24

Compound 15

BCL6

CRBN

< 1 μM/84% at 1 μM

In vitro cellular probe

42

(continued on next page) 1558

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

Table 1 (continued) PROTAC

Structure

Name

Target (POI)

E3 Ligase

DC50/Dmax

Cellular/in vivo

Ref

25

JH-XI-10–02

CDK8

CRBN

1 μM/significant deg at 1 μM

In vitro

43

26

THAL-SNS-032

CDK9

CRBN

< 250 nM/ > 95%

In vitro

44

27

Compound 3

CDK9

CRBN

10 μM/ > 65% at 20 μM

In vitro

45

28

Compound D

PI3K

CRBN

< 25 μM/ > 60% at 50 μM

In vitro

46

29

MT-802

BTK

CRBN

9.1 nM/ > 99% at 100 nM

In vitro

47

30

P13I

BTK

CRBN

> 6- < 12 nM/ > 94% at 500 nM

In vitro

48

31

Compound 10

BTK

CRBN

1.1 nM /87% at 300 nM

In vivo probe (SC dose)

49

32

TL13-112

ALK

CRBN

10 nM/ > 99% at 0.5 μM

In vitro

50

33

Compound 9C

HDAC6

CRBN

34 nM/70% at 370 nM

In vitro

51

34

Compound 12

Sirtuin

CRBN

0.2–1 μM/90% at 5 μM

In vitro

52

35

GSK983

PCAF/GCN5

CRBN

1.5 nM/97% (PCAF) 3 nM 81% (GCN5)

In vitro

53

36

CCT367766

Pirin

CRBN

1–2.5 nM/ > 99%

In vitro

54

37

dTAG13

FKBP12F36V

CRBN

< 100 nM/82%

In vivo (IP)

55

38

RC32

FKBP12

CRBN

0.3 nM/ > 95%

In vivo (oral, mice)

56

1559

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

Table 2 In silico metrics for selected PROTACs.

Algorithms for calculation of cLogP,65 cLogD, and nRotB are referenced. A bond is defined as rotatable if it is single and acyclic with neither atom being metal or bonded to metal, and both atoms must be bonded to at least one other non-terminal heavy atom. Color coding is not intended to be predictive of absorption, but instead was assigned to display a range of properties. Colors are defined as follows. Red: MW > 1000; cLogP > 9; HBD ≥ 5; HBA > 17; PSA > 250; Rot B > 20; Nrule-of-5 ≥ 3; AB-MPS > 20. Yellow/Orange: MW 700–1000; cLogP 6–9; HBD 4; HBA 13–17; PSA 200–250; Rot B 15–20; Nrule-of-5 = 2; AB-MPS 15–20. Green: MW < 700; cLogP < 6; HBD ≤ 3; HBA ≤ 12; PSA < 200; Rot B < 15; Nrule-of-5 ≤ 1; AB-MPS < 15. 1560

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

are less flexible than others (e.g. nRotB and hydrogen bond donor count). Although a recent analysis challenges the utility of these types of metrics,63 they offer value in that they provide guidelines by which medicinal chemists may prioritize the design and synthesis of new compounds with a greater success of oral absorption. To date, in vivo studies reported with PROTACs in peer-reviewed literature have typically been performed through parenteral delivery rather than oral administration. An ability to orally administer PROTACs would increase the attractiveness of this new modality as therapeutic agents. To explore the potential of PROTACs for oral bioavailability, we evaluated several physicochemical descriptors of PROTACS associated with compound permeability/absorption including molecular weights (MW), lipophilicities (cLogP, cLogD), Hbond donors (HBDs), H-bond acceptors (HBAs), polar surface areas (PSAs), nRotB, the number of violations of Lipinski’s rule-of-5 (Nrule-of64 and AB-MPS values (Table 2). Importantly, these PROTACs were 5), not necessarily intended to be dosed orally, rather they were either cellular tools or in vivo tools intended to validate PROTAC biology. Nevertheless, as optimized tools, the compounds in Table 2 collectively form a representative set of potent PROTACs that could be a starting point for the pursuit of orally bioavailable PROTACs. MDM2-PROTACs exhibit the highest MW and are the most lipophilic group of PROTACs. They also possess the highest PSAs compared to the other E3 sub-classes. Furthermore, they have 3 aromatic rings in the MDM2 ligand moiety alone and together with the high lipophilicity and nRotB (Table 3), their AB-MPS and Nrule-of-5 suggest that oral absorption will be a high hurdle. Similarly, IAP-PROTACs exhibit high MWs, high lipophilicities, and high nRotBs, also highlighting the challenge of achieving exposure through oral dosing of this PROTAC subclass. VHL-PROTACs fare better in an analysis of their properties although on average they still possess high MWs and nRotBs. A few VHL-PROTACs (e.g. 10, 12 and 18) have reduced HBDs and/or relatively low PSAs, but they are still high in terms of overall Nrule-of-5 and AB-MPS scores. PROTAC 18, with its new VHL connection vector, offers an advantage in terms of reduced PSA and HBD/HBA counts. Further optimization of this sub-series to reduce MW and nRotB may help move it into even more promising chemical space. On average, however, the AB-MPS and Nrule-of-5 scores for VHL-PROTACs are still high, suggesting a low likelihood for oral absorption. Due to the distinct starting properties of their E3 warhead, CRBN-PROTACs are in a more suitable chemical space with respect to oral absorption (Table 3). Their MWs can drop below 700 and they have reasonably drug-like lipophilicities and reduced HBD/nRotB counts. Compounds 23 and 27, for example, are in chemical space more likely to result in orally absorbable

Fig. 2. Upper boundary limits of physicochemical properties of Ro5 compounds (Lipinski and Veber) and bRo5 compounds (Kihlberg, AbbVie) plotted with the mean property values for PROTACs from Table 2 with various E3 warheads. The mean AB-MPS scores are included for comparison.

compounds as evidenced by their overall improved physicochemical properties. The averaged values for in silico descriptors of MDM2-, IAP-, VHLand CRBN-PROTACs can be summarized on a radar plot similar to that reported by Kihlberg, et al. where we compare the PROTAC properties to the upper boundary limits for sets of Ro5 and bRo5 compounds (Fig. 2). MDM2- and IAP-PROTACs are either at or beyond the upper limits for acceptable oral absorption defined by bRo5 descriptors. VHLPROTACs are closer, but on average still suffer from nRotB higher than the upper boundary and a higher number of HBDs compared to the other E3 warheads. On average, the CRBN PROTACs come closer to ‘drug-like’ space with their average HBD and cLogP values falling within the Ro5 boundaries. All these series suffer from high RotB and HBA counts that are either beyond or at the upper limits of these analyses, suggesting at least two areas where medicinal chemists can focus to further improve chances for oral absorption of PROTACs. Importantly, the list of PROTACs in Tables 1/2 contains outliers at the outer limits of absorbable space (e.g. 6, 14, 37) as well as within the boundaries of potentially absorbable space (23, 27). An area that merits further exploration for PROTACs is the relationship between secondary structure and ADME properties. These molecules are moderately-sized and owing to their chimeric structures, they might be expected to adopt different solution conformations with varying degrees of flexibility. Despite possessing high nRotBs, their

Table 3 Representative E3 ligase ligands and their calculated properties.

Parameter

Idasanutlin Amide (MDM2 ligand)

LCL-161 (IAP ligand)

HIF1α binder (VHL ligand)

Thalidomide (CRBN ligand)

MW cLogP HBD HBA nRotB PSA Ar Rings

630 6.2 3 7 11 108 3

500 3.6 2 7 10 95 2

472 2.0 3 8 10 118 2

258 0.53 1 6 1 89 1

1561

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

secondary structure may also confer a degree of rigidity that could influence their propensity for metabolism and/or permeability. For example, recent reports suggest that a degree of flexibility could result in chameleonic effects to molecules in the bRo5 space, allowing them to display different degrees of polarity according to their environment and consequently potential advantages from an absorption perspective.66 Various analytical methods exist for monitoring these phenomenon including the measurement of exposed polar surface areas (EPSA),67 new experimental measures of lipophilicity,68 and/or measurement of NMR solution conformations in various media.69 Future work focused on better understanding of the relationships between secondary structure and absorption/metabolism will help inform PROTAC design strategies to successfully meet the challenge of oral absorption. Another parameter that has a significant impact on absorption and in vitro assay quality is solubility. Without adequate solubility in the gut, oral absorption will be challenging. Moreover, poor solubility in assay media can affect the quality of assay data due to compound precipitation and lower solution concentrations than expected. The CRBN ligand thalidomide was reported to have poor aqueous solubility at a range of pHs which may also confer solubility challenges to their corresponding PROTACs.70 Little data has been reported to date for PROTAC solubilities; however, it is reassuring to note that both PBS (phosphate-buffered saline) and simulated intestinal fluid solubilities for a series of BRD4 PROTACs containing a triazole in the linker was comparable to a series of matched pair analogs with simple alkyl/alkoxy linkers.27 All these PROTACs exhibited adequate solubility to support gut absorption at drug-like doses. Beyond solubility, other physicochemical properties such as chemical stability and non-specific binding to labware and/or protein in media could also impact the ability to accurately interpret assay data from PROTACs. For example, thalidomide has been reported to be configurationally unstable through facile racemization at the glutarimide moiety,71 and the two enantiomers have been reported to have different biological activities.72 Additionally, the phthalimide and glutarimide functionalities on CRBN ligands have been reported to be hydrolytically unstable due to hydrolysis at either of the imide moieties.29,45,73,74 Unless issues such as chemical stability and/or nonspecific binding are adequately addressed, in vitro assay data may be compromised and this in turn may impact the ability to interpret PK/PD data or to make human ADME predictions with confidence. Physicochemical descriptors like those in Table 2 are helpful for contextualizing risks around oral absorption and certain physicochemical properties, but there may be additional challenges that lie ahead for PROTACs beyond absorption.75 Importantly, oral bioavailability relies not only on absorption, but also on metabolism in both the gut and the liver.76 Most PROTACs are relatively lipophilic with cLogPs greater than 4 and consequently may suffer from higher oxidative metabolism than polar drugs. Additionally, linkers with linear aliphatic chains and/or ether chains are expected to carry risk for oxidative metabolism depending on the ease of linker access to oxidizing enzyme catalytic sites. Consequently, to achieve adequate oral bioavailability for PROTACs, a progression cascade must also focus on empirical reduction of clearance, typically by reduction of unbound clearance in liver microsome and/or hepatocytes with in vitro assays prior to progression into in vivo PK studies.

POI-PROTAC or E3 ligase-PROTAC binary complexes instead of the desired POI-PROTAC-E3 ligase ternary complex.29,31 For POIs with long resynthesis times where the efficacy/pharmacodynamic (PD) effect of a PROTAC may be Cmax driven, the “hook” effect can be attenuated through management of the pharmacokinetic profile of the PROTAC. For POIs with shorter resynthesis times, however, the rate of PROTACmediated degradation will compete with POI resynthesis, and a steadystate concentration of PROTAC will be needed to maintain the desired PD effect. It is common for systemic drug concentrations to fluctuate, however, and so if the “hook” effect translates in vivo, reduced protein degradation may be observed at high drug concentrations for POIs with shorter resynthesis times. Key pharmacological parameters that contribute to an in vivo “hook” effect likely include the kinetics and binding affinity to the designated E3 ligase/POI ligand, the cooperativity in ternary complex formation and the expression level of the E3 ligase. Accordingly, the choices of E3 ligase, E3 ligase ligands and linkers with optimal cooperativity are important to attenuate this effect. Additionally, PROTACs with long plasma half-lives (i.e. low peak-totrough ratios) would be desirable to mitigate an in vivo “hook” effect in this scenario. An additional challenge for PROTACs to achieve their optimal in vivo effect is related to the necessity of a ternary complex to afford degradation. PROTACs will form metabolites in vivo that may have the potential to maintain relevant systemic concentrations. Though these metabolites may lose the ability to form efficient ternary complexes, they may be expected to retain binding affinity towards either the POI or the E3 ligase. Consequently, they could have the potential to adversely impact PROTAC-mediated degradation by competitively forming binary complexes with either the POI or the E3 ligase. Alternatively, a metabolite may also have safety/toxicity risks thus limiting the therapeutic benefit of the PROTAC (although this is not unique to PROTACs). Consequently, a key strategy to mitigate risks posed by metabolites will be to monitor for them and minimize their formation as much as possible during lead optimization. To date, PROTACs research has mainly focused on a small number of E3 ligases, such as CRBN and VHL. Current literature suggests these E3 ligases are ubiquitously expressed in humans, so target-mediated drug disposition may be less of a concern for these ligases. Nevertheless, as researchers investigate alternative E3 ligases with tissue specific expression levels, the relative expression across a variety of tissues need to be understood from a pharmacology/toxicology perspective as well as for ADME considerations. Beyond ADME challenges, PROTACs also move drug discovery efforts into uncharted territories with regards to drug safety. Many of the PROTACs reported above exhibit excellent degradation selectivity for their desired POIs, but little literature is available for their general offtarget activity profiles in large panels of receptors, enzymes, and ion channels. With their relatively high lipophilicities (cLogPs > 4, Table 2), PROTACs may have some secondary pharmacology due to offtarget activities, increasing the risk of dose-limiting side effects in a clinical setting.77 Additionally, the necessary binding affinity of PROTACs to the designated E3 ligase could also be a liability for drug safety concerns. For example, early evidence suggests that some CRBN-PROTACs may retain their pharmacology at inducing zinc-finger family protein degradation and their associated pharmacologies.49 Finally, while PROTACs have the potential to achieve superior selectivity in inducing POI degradation than small molecule functional inhibitors, concerns remain that off-target protein degradation could cause collateral damage to normal tissue, the so-called bystander effect. Moreover, any off-target receptor pharmacology of the PROTAC might also be expected to translate to a potential dose-limiting effect in the clinic. Facing these challenges, the general drug discovery strategy of pursuing highly potent and specific drug molecules still holds true. In the case of PROTACs, this means that choosing selective and potent POI ligands, matching them with an effective E3 ligase ligand and optimizing the linkers to maximize cooperativity, will ultimately lead to the

Future challenges Through optimization of both absorption and metabolism it is likely that orally bioavailable PROTACs will be designed giving this modality one of the key advantages of small molecules. Nevertheless, some unanswered questions and key challenges lie ahead as PROTACs progress into clinical trials with the aim of delivering optimal disease treatment for patients. One clear complexity unique to PROTACs is the so-called “hook” effect, where in vitro cellular target protein degradation is attenuated at higher PROTAC concentrations likely due to competitive formation of 1562

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al.

identification of highly potent and selective degraders with the best chances of progression to a drug.

20. Itoh Y, Ishikawa M, Naito M, Hashimoto Y. Protein knockdown using methyl bestatin-ligand hybrid molecules: design and synthesis of inducers of ubiquitinationmediated degradation of cellular retinoic acid-binding proteins. J Am Chem Soc. 2010;132(16):5820–5826. 21. Ohoka N, Okuhira K, Ito M, et al. In vivo knockdown of pathogenic proteins via specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs). J Biol Chem. 2017;292(11):4556–4570. 22. Shibata N, Nagai K, Morita Y, et al. Development of protein degradation inducers of androgen receptor by conjugation of androgen receptor ligands and inhibitor of apoptosis protein ligands. J Med Chem. 2018;61(2):543–575. 23. Ohoka N, Morita Y, Nagai K, et al. Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor alpha degradation. J Biol Chem. 2018;293(18):6776–6790. 24. Willam C, Masson N, Tian YM, et al. Peptide blockade of HIFalpha degradation modulates cellular metabolism and angiogenesis. Proc Natl Acad Sci USA. 2002;99(16):10423–10428. 25. Buckley DL, Van Molle I, Gareiss PC, et al. Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1alpha interaction. J Am Chem Soc. 2012;134(10):4465–4468. 26. Zengerle M, Chan KH, Ciulli A. Selective Small molecule induced degradation of the BET bromodomain protein BRD4. ACS Chem Biol. 2015;10(8):1770–1777. 27. Wurz RP, Dellamaggiore K, Dou H, et al. A “Click Chemistry Platform” for the rapid synthesis of bispecific molecules for inducing protein degradation. J Med Chem. 2018;61(2):453–461. 28. Raina K, Lu J, Qian Y, et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc Natl Acad Sci USA. 2016;113(26):7124–7129. 29. Gadd MS, Testa A, Lucas X, et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol. 2017;13(5):514–521. 30. Chan KH, Zengerle M, Testa A, Ciulli A. Impact of target warhead and linkage vector on inducing protein degradation: comparison of bromodomain and extra-terminal (BET) degraders derived from triazolodiazepine (JQ1) and tetrahydroquinoline (IBET726) BET inhibitor scaffolds. J Med Chem. 2018;61(2):504–513. 31. Bondeson DP, Mares A, Smith IE, et al. Catalytic in vivo protein knockdown by smallmolecule PROTACs. Nat Chem Biol. 2015;11(8):611–617. 32. Lai AC, Toure M, Hellerschmied D, et al. Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew Chem Int Ed Engl. 2016;55(2):807–810. 33. Huang HT, Dobrovolsky D, Paulk J, et al. A chemoproteomic approach to query the degradable kinome using a multi-kinase degrader. Cell Chem Biol. 2018;25(1) 88-99 e86. 34. Cromm PM, Samarasinghe KTG, Hines J, Crews CM. Addressing kinase-independent functions of fak via PROTAC-mediated degradation. J Am Chem Soc. 2018. 35. Crew AP, Raina K, Dong H, et al. Identification and characterization of von hippellindau-recruiting proteolysis targeting chimeras (PROTACs) of TANK-binding kinase 1. J Med Chem. 2018;61(2):583–598. 36. Zoppi V, Hughes SJ, Maniaci C, et al. Iterative design and optimization of initially inactive Proteolysis Targeting Chimeras (PROTACs) identify VZ185 as a potent, fast and selective von Hippel-Lindau (VHL)-based dual degrader probe of BRD9 and BRD7. J Med Chem. 2018. 37. Han X, Wang C, Qin C, et al. Discovery of ARD-69 as a highly potent proteolysis targeting chimera (PROTAC) degrader of androgen receptor (AR) for the treatment of prostate cancer. J Med Chem. 2019. 38. Winter GE, Buckley DL, Paulk J, et al. Drug development Phthalimide conjugation as a strategy for in vivo target protein degradation. Science. 2015;348(6241):1376–1381. 39. Lu J, Qian Y, Altieri M, et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem Biol. 2015;22(6):755–763. 40. Zhou B, Hu J, Xu F, et al. Discovery of a small-molecule degrader of bromodomain and extra-terminal (BET) proteins with picomolar cellular potencies and capable of achieving tumor regression. J Med Chem. 2018;61(2):462–481. 41. Qin C, Hu Y, Zhou B, et al. Discovery of QCA570 as an exceptionally potent and efficacious proteolysis targeting chimera (PROTAC) degrader of the bromodomain and extra-terminal (BET) proteins capable of inducing complete and durable tumor regression. J Med Chem. 2018;61(15):6685–6704. 42. McCoull W, Cheung T, Anderson E, et al. Development of a novel B-cell lymphoma 6 (BCL6) PROTAC To provide insight into small molecule targeting of BCL6. ACS Chem Biol. 2018;13(11):3131–3141. 43. Hatcher JM, Wang ES, Johannessen L, Kwiatkowski N, Sim T, Gray NS. Development of highly potent and selective steroidal inhibitors and degraders of CDK8. ACS Med Chem Lett. 2018;9(6):540–545. 44. Olson CM, Jiang B, Erb MA, et al. Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation. Nat Chem Biol. 2018;14(2):163–170. 45. Robb CM, Contreras JI, Kour S, et al. Chemically induced degradation of CDK9 by a proteolysis targeting chimera (PROTAC). Chem Commun (Camb). 2017;53(54):7577–7580. 46. Li W, Gao C, Zhao L, Yuan Z, Chen Y, Jiang Y. Phthalimide conjugations for the degradation of oncogenic PI3K. Eur J Med Chem. 2018;151:237–247. 47. Buhimschi AD, Armstrong HA, Toure M, et al. Targeting the C481S ibrutinib-resistance mutation in Bruton’s tyrosine kinase using PROTAC-mediated degradation. Biochemistry. 2018;57(26):3564–3575. 48. Sun Y, Zhao X, Ding N, et al. PROTAC-induced BTK degradation as a novel therapy for mutated BTK C481S induced ibrutinib-resistant B-cell malignancies. Cell Res. 2018;28(7):779–781. 49. Zorba A, Nguyen C, Xu Y, et al. Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc Natl Acad Sci USA. 2018;115(31):E7285–E7292. 50. Powell CE, Gao Y, Tan L, et al. Chemically induced degradation of anaplastic

Conclusion Though a relatively new modality, PROTACs have progressed rapidly through drug discovery pipelines and an androgen receptor degrader has recently entered into human clinical trials as an orally dosed PROTAC – thus underscoring the likelihood that this new modality will yield oral marketed drugs.78 Furthermore, they can successfully degrade a variety of protein types suggesting broad scope to treat a variety of diseases across multiple therapeutic areas. Nevertheless, this new modality will face complexities to achieve and understand oral bioavailability, PK/PD/efficacy relationships,79 and distribution, metabolism, and toxicity challenges. As further progress is reported in ADME optimization, however, interest is anticipated to continue to grow. In the coming years, a more detailed understanding of in vivo efficacy and safety studies from orally dosed PROTACs will help shed light on their future potential and breadth. Although challenges lie ahead, PROTACs represent a new area to further expand the druggable genome and as an emerging new therapeutic modality they offer the exciting potential to provide medical benefits to patients in need. Acknowledgements The authors wish to thank Dr. Sameer Kawatkar for his help in assembling a convenient database of calculated properties which was used to determine the data in Table 2. The authors also thank several people for their helpful comments in reviewing this manuscript including Drs. Andrew Pike, Dermot McGinnity, James Scott, Kurt Pike, Neil Grimster, and Beth Williamson. References 1. Ecker DM, Jones SD, Levine HL. The therapeutic monoclonal antibody market. MAbs. 2015;7(1):9–14. 2. Geary RS, Norris D, Yu R, Bennett CF. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev. 2015;87:46–51. 3. Wittrup A, Lieberman J. Knocking down disease: a progress report on siRNA therapeutics. Nat Rev Genet. 2015;16(9):543–552. 4. Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. Drug Discov Today. 2015;20(1):122–128. 5. Wang T, Chen C, Larcher LM, Barrero RA, Veedu RN. Three decades of nucleic acid aptamer technologies: Lessons learned, progress and opportunities on aptamer development. Biotechnol Adv. 2019;37(1):28–50. 6. Kaczmarek JC, Kowalski PS, Anderson DG. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med. 2017;9(1):60. 7. Xiao-Jie L, Hui-Ying X, Zun-Ping K, Jin-Lian C, Li-Juan J. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet. 2015;52(5):289–296. 8. Hopkins AL, Groom CR. The druggable genome. Nat Rev Drug Discov. 2002;1(9):727–730. 9. Burslem GM, Smith BE, Lai AC, et al. The advantages of targeted protein degradation over inhibition: An RTK case study. Cell Chem Biol. 2018;25(1) 67-77 e63. 10. Ottis P, Crews CM. Proteolysis-targeting chimeras: induced protein degradation as a therapeutic strategy. ACS Chem Biol. 2017;12(4):892–898. 11. Lai AC, Crews CM. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev Drug Discov. 2017;16(2):101–114. 12. Burslem GM, Crews CM. Small-Molecule Modulation of Protein Homeostasis. Chem Rev. 2017;117(17):11269–11301. 13. Gu S, Cui D, Chen X, Xiong X, Zhao Y. PROTACs: an emerging targeting technique for protein degradation in drug discovery. BioEssays. 2018;40(4):e1700247. 14. Churcher I. Protac-induced protein degradation in drug discovery: breaking the rules or just making new ones? J Med Chem. 2018;61(2):444–452. 15. Zou Y, Ma D, Wang Y. The PROTAC technology in drug development. Cell Biochem Funct. 2019. 16. An S, Fu L. Small-molecule PROTACs: An emerging and promising approach for the development of targeted therapy drugs. EBioMedicine. 2018;36:553–562. 17. Zou Y, Ma D, Wang Y. The PROTAC technology in drug development. Cell Biochem Funct. 2019;37(1):21–30. 18. Schneekloth AR, Pucheault M, Tae HS, Crews CM. Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics. Bioorg Med Chem Lett. 2008;18(22):5904–5908. 19. Hines J, Lartigue S, Dong H, Qian Y, Crews CM. MDM2-recruiting PROTAC offers superior, synergistic antiproliferative activity via simultaneous degradation of BRD4 and stabilization of p53. Cancer Res. 2019;79(1):251–262.

1563

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1555–1564

S.D. Edmondson, et al. lymphoma kinase (ALK). J Med Chem. 2018;61(9):4249–4255. 51. Yang K, Song Y, Xie H, et al. Development of the first small molecule histone deacetylase 6 (HDAC6) degraders. Bioorg Med Chem Lett. 2018;28(14):2493–2497. 52. Schiedel M, Herp D, Hammelmann S, et al. Chemically induced degradation of sirtuin 2 (Sirt2) by a proteolysis targeting chimera (PROTAC) based on sirtuin rearranging ligands (SirReals). J Med Chem. 2018;61(2):482–491. 53. Bassi ZI, Fillmore MC, Miah AH, et al. Modulating PCAF/GCN5 immune cell function through a PROTAC approach. ACS Chem Biol. 2018;13(10):2862–2867. 54. Chessum NEA, Sharp SY, Caldwell JJ, et al. Demonstrating in-cell target engagement using a pirin protein degradation probe (CCT367766). J Med Chem. 2018;61(3):918–933. 55. Nabet B, Roberts JM, Buckley DL, et al. The dTAG system for immediate and targetspecific protein degradation. Nat Chem Biol. 2018;14(5):431–441. 56. Sun X, Wang J, Yao X, et al. A chemical approach for global protein knockdown from mice to non-human primates. Cell Discov. 2019;5:10. 57. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001;46(1–3):3–26. 58. Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD. Molecular properties that influence the oral bioavailability of drug candidates. J Med Chem. 2002;45(12):2615–2623. 59. Doak BC, Over B, Giordanetto F, Kihlberg J. Oral druggable space beyond the rule of 5: insights from drugs and clinical candidates. Chem Biol. 2014;21(9):1115–1142. 60. Doak BC, Zheng J, Dobritzsch D, Kihlberg J. How beyond rule of 5 drugs and clinical candidates bind to their targets. J Med Chem. 2016;59(6):2312–2327. 61. DeGoey DA, Chen HJ, Cox PB, Wendt MD. Beyond the rule of 5: lessons learned from AbbVie's drugs and compound collection. J Med Chem. 2018;61(7):2636–2651. 62. Poongavanam V, Doak BC, Kihlberg J. Opportunities and guidelines for discovery of orally absorbed drugs in beyond rule of 5 space. Curr Opin Chem Biol. 2018;44:23–29. 63. Shultz MD. Two decades under the influence of the rule of five and the changing properties of approved oral drugs. J Med Chem. 2019;62(4):1701–1714. 64. Hou T, Wang J, Zhang W, Xu X. ADME evaluation in drug discovery. 7. Prediction of oral absorption by correlation and classification. J Chem Inf Model. 2007;47(1):208–218. 65. Wildman SA, Crippen GM. Prediction of physicochemical parameters by atomic contributions. J Chem Inf Comput Sci. 1999;39(5):868–873.

66. Rossi Sebastiano M, Doak BC, Backlund M, et al. Impact of dynamically exposed polarity on permeability and solubility of chameleonic drugs beyond the rule of 5. J Med Chem. 2018;61(9):4189–4202. 67. Goetz GH, Philippe L, Shapiro MJ. EPSA: a novel supercritical fluid chromatography technique enabling the design of permeable cyclic peptides. ACS Med Chem Lett. 2014;5(10):1167–1172. 68. Ermondi G, Vallaro M, Goetz G, Shalaeva M, Caron G. Experimental lipophilicity for beyond Rule of 5 compounds. Future Drug Disc. 2019 eISSN 2631–3316. 69. Over B, Matsson P, Tyrchan C, et al. Structural and conformational determinants of macrocycle cell permeability. Nat Chem Biol. 2016;12(12):1065–1074. 70. Krenn M, Gamcsik MP, Vogelsang GB, Colvin OM, Leong KW. Improvements in solubility and stability of thalidomide upon complexation with hydroxypropyl-betacyclodextrin. J Pharm Sci. 1992;81(7):685–689. 71. Ogino Y, Tanaka M, Shimozawa T, Asahi T. LC-MS/MS and chiroptical spectroscopic analyses of multidimensional metabolic systems of chiral thalidomide and its derivatives. Chirality. 2017;29(6):282–293. 72. Mori T, Ito T, Liu S, et al. Structural basis of thalidomide enantiomer binding to cereblon. Sci Rep. 2018;8(1):1294. 73. Reist M, Carrupt PA, Francotte E, Testa B. Chiral inversion and hydrolysis of thalidomide: mechanisms and catalysis by bases and serum albumin, and chiral stability of teratogenic metabolites. Chem Res Toxicol. 1998;11(12):1521–1528. 74. Hoffmann M, Kasserra C, Reyes J, et al. Absorption, metabolism and excretion of [14C]pomalidomide in humans following oral administration. Cancer Chemother Pharmacol. 2013;71(2):489–501. 75. Scott JS, Waring MJ. Practical application of ligand efficiency metrics in lead optimisation. Bioorg Med Chem. 2018;26(11):3006–3015. 76. El-Kattan A, Varma M. Oral absorption, intestinal metabolism and human oral bioavailability. Topics on Drug Metab. 2012. 77. Leeson PD, Springthorpe B. The influence of drug-like concepts on decision-making in medicinal chemistry. Nat Rev Drug Discov. 2007;6(11):881–890. 78. Mullard A. First targeted protein degrader hits the clinic. Nat Rev Drug Discov. 2019;18:237–239. 79. Watt GF, Scott-Stevens P, Gaohua L. Targeted protein degradation in vivo with Proteolysis Targeting Chimeras: Current status and future considerations. Drug Discov Today: Technol. 2019. https://doi.org/10.1016/j.ddtec.2019.02.005.

1564