[PROTAC image courtesy of Wikipedia]

PROteolysis TArgeting Chimera (PROTAC)* drugs emerged in 2001 as a novel therapeutic option for treating previously untreatable diseases. To date, no PROTAC drugs have earned U.S. FDA approval, but the technology has opened up a promising new chapter in drug discovery. PROTACs are being widely explored across both industry and academia and have expanded from treating cancer to immune disorders, neurodegenerative diseases, viral infections, etc.

A PROTAC molecule consists of three components: a ligand binding to a target protein, a ligand binding to E3 ligase and a linker. Unlike conventional small molecules that block target proteins via inhibition, PROTACs bind specifically to target proteins and induce their degradation by leveraging the ubiquitin-proteasome system (UPS). PROTACs’ binding process is referred to as a “chemical knockdown” approach.

PROTACs boast several advantages over conventional small molecules, including:

  • High potency: PROTACs are effective at lower concentrations. Their efficacy is sustained for a longer time since the restoration of target protein function requires the resynthesis of the target protein.
  • Target “undruggable” diseases: Unlike small molecule inhibitors, PROTACs’ binding to drug targets does not always require an ideal binding site to inhibit the activity of target protein. Some disease-causing dysfunctional proteins, including nuclear receptors, transcription factors and skeleton proteins, can be targeted by PROTACs but not routine inhibitors.

Despite PROTACs’ clear advantages, significant questions remain around their DMPK characteristics. PROTACs display promising pharmacological activity, but their poor pharmacokinetic behavior often thwarted research efforts.

In this second article in our two-part series on PROTACs, we will explore some of these DMPK challenges — focusing specifically on improving oral bioavailability — and what it means for drug sponsors and developers.

[Related: Tackling the DMPK challenges of developing PROTAC drugs]

The problems with PROTACs

Most discussions of PROTAC challenges start by citing the Lipinksi Rule of Five (Ro5). A set of rules to determine “druggability” guidelines for new compounds, the Ro5 says compounds are more likely to have poor absorption or permeation when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight is greater than 500, the calculated log P is great than 5 and rotatable bonds are over 10. Due to their structural features, PROTACs typically belong to the beyond Ro5 chemical space. PROTACs’ molecular weight is generally between 700 and 1200 Da, making them more difficult to meet other criteria.

Low oral bioavailability

PROTAC molecules have high molecular weight, poor solubility and low permeability,

making it challenging to improve oral bioavailability in vivo. To do so, developers must optimize PROTACs’ physicochemical and pharmacokinetic properties. Optimization of this kind may impact the drug’s efficacy, but the aim here is to explore options to improve pharmacokinetic properties.

Improving PROTACs’ oral bioavailability

1. Administer with food

Oral medications need to be dissolved before they are absorbed in the intestine, but PROTACs usually have very poor aqueous solubility. Since oral absorption of drugs occurs in the intestine, the simulated solution must mimic the real environment of intestinal absorption. Recent research has found that PROTACs showed improved solubility in a biorelevant buffer such as FaSSIF/FeSSIF. This finding suggests that the in vivo pharmacokinetics of PROTACs may obtain a better in vivo drug exposure after the patient has eaten. The clinical trial design of ARV-110 and ARV-471 also disclosed that the Phase I clinical administration mode of these two PROTAC molecules was “once daily with food.”

2. Improve metabolic stability

When being absorbed by the intestine, the compound will be metabolized by the liver or intestine as the route to enter systemic circulation. This is called “first-pass” metabolism, which limits the oral absorption of most drugs. To improve oral bioavailability, improving metabolic stability is another strategy that drug developers can take to enhance the success of their PROTAC development. Structurally, modifying two ligands is restricted by their target proteins, so the linker part appears to be more flexible for optimizing PROTACs. Multiple strategies, including changing linker length, changing the linker’s anchor point, using cyclic linkers, and altering the linker’s attachment site, have been investigated to improve metabolic stability.

3. Improve cellular permeability

Optimizing the linker structure can also improve cellular permeability. The oral absorption of drugs needs to pass through the membrane barrier of the small intestine. To achieve intracellular protein degradation, PROTACs also need to enter target cells. Research has shown that replacing a PEG linker with a 1,4-disubstituted phenyl ring significantly improves PROTAC cellular permeability. Multiple amide motifs in the liker structure should be avoided to preserve permeability. Instead, inserting basic nitrogen into aromatic rings or alkyl linkers has been proved useful in improving solubility.

4. Choose smaller E3 ligands

The properties of PROTAC are closely related to the types of E3 ligases. It is reported that E3 ligases applied to PROTACs mainly include CRBN, VHL, IAP and MDM2, among them, CRBN and VHL are the most frequently used. VHL-targeted PROTACs are unlikely to have high oral exposure. But CRBN-targeted PROTACs, which have smaller molecular weight, are more “oral drug-like.” Given two examples, the two PROTAC molecules ARV-110 and ARV-471 entering clinical phase II are the ligands of CRBN E3 ligase. Finding new E3 ligands with smaller molecular weight is worth exploring.

5. Introduce intramolecular hydrogen bonds

PROTACs generally have high polarity and many rotatable bonds. Such a structure does not allow passage across the cell membrane’s lipid bilayer. Recent research found that formation of intramolecular hydrogen bonds will facilitate cell permeability of PROTACs by reducing their molecular size and polarity. As the research illustrated, an original strip-type molecule was transformed into a “ball” form. The most challenging component of the transformation is introducing intramolecular hydrogen bonds in the design of PROTAC structure. It is a shortcut, but it may also require a little luck.

6. Use a prodrug strategy

Prodrug is a common approach to improving a drug’s oral bioavailability. A prodrug is obtained by modifying a pharmacologically active compound. Prodrugs themselves have little to no activity and will transform in vivo to release the active metabolite by enzyme catalysis. It is yet to be seen if using prodrugs will become a successful tactic for PROTACs in practice. One potential disadvantage is that using a prodrug may further increase the PROTACs’ molecular weight.

Nevertheless, chemists are trying to minimize concerns. Scientists have designed a prodrug from a PROTAC compound by adding a lipophilic group to the CRBN ligand. Results showed a significant increase in the PROTAC’s bioavailability. Since it is a CRBN-based prodrug, this design can be used for other PROTACs with similar E3 ligands.

7. Use molecular glues

PROTACs consist of three parts: two separate ligands and a linker. Inevitably, such a heterobifunctional molecule will have relatively large molecular weight. Chemists continue to explore new strategies to reduce the molecular weight to make PROTACs more “drug-like.” Molecular glues are considered “compact molecules” that lack a linker and can trigger a ternary complex like that of PROTACs. For these reasons, molecular glues are showing promise as new alternatives.

A final word on PROTACs

Most conventional small molecules rely on an ‘occupancy-driven’ mechanism of action (MOA). This means the drug’s efficacy depends on the sustained binding of a molecule to the target. High doses are usually required to achieve adequate target engagement, which can lead to off-target toxicity. For PROTAC drugs, their MOA is “event-driven.” The binding of a PROTAC to a drug target will trigger ubiquitination and subsequent degradation of the target protein.

PROTACs are providing unprecedented therapeutic options, including the potential to target “undruggable” diseases. Despite industry-wide optimism around some late-phase clinical studies, drug sponsors and developers still face the challenge of optimizing PROTACs’ pharmacokinetic behavior without undermining the drug’s efficacy. Greater collaboration between academia, drug developers and lab testing partners will be necessary to solve the PROTAC puzzle and realize their full potential.

*PROTAC® is a registered trademark of Arvinas. In this article, PROTAC refers explicitly to the abbreviation of PROteolysis TArgeting Chimera as therapeutic modalities.

Dr. Liping Ma, Ph.D. of Pharmaceutical Analysis, an expert in PROTAC pharmacokinetic evaluation, is now a senior study director in the DMPK Service Department of WuXi AppTec. She has over 10 years of research experience in preclinical and clinical pharmacokinetics and extensive experience in the preclinical development of drugs. She successfully supported more than 20 new drug IND applications globally.