7 strategies to improve oral bioavailability of PROTAC drugs

preface

Compared with traditional small molecule drugs, protein degradation targeting chimera, or Proteolysis-Targeting Chimeras (referred to as "PROTAC" in context), has two major advantages in terms of mechanism of action:

Highly efficient:

The process of PROTAC degradation of target proteins is similar to the action of catalysts and is effective
at low drug concentrations.
It directly degrades the target protein, and the restoration of the target protein's function requires the resynthesis of the protein so that its efficacy lasts longer
.

Ability to target "undruggable" targets:

Unlike small molecule inhibitors, the binding of PROTAC to drug targets does not always require an ideal active binding site to inhibit the function of the target protein, which can bind to any site
.
For example, some disease-causing dysfunctional proteins, including nuclear receptors, transcription factors, skeleton proteins, etc.
, are difficult to be inhibited by conventional small molecule drugs due to the lack of typical active sites, but can be targeted by PROTAC
.

Despite the obvious advantages of PROTAC, there are still some problems
with its DMPK nature.
PROTACs with good pharmacological activity tend to disappoint
due to their poor pharmacokinetic properties.
In this article, we will explore the challenges of DMPK and provide some strategies to improve oral bioavailability, hoping to bring new ideas
to PROTAC developers.

The development of PROTAC technology began in 2001, and the period from 2001 to 2018 is considered the yesterday
of PROTAC development.
During this time, the mechanism of action of PROTAC was elucidated, the E3 ligase and ligand used for over-the-counter drugs were discovered, and the preclinical efficacy was confirmed
.
In 2019, the world's first and second PROTAC molecules entered phase I clinical trials, which became a landmark event
.
In 2020, the two PROTAC molecules that entered the clinic disclosed clinical data, which brought great hope
for the research and development of such molecules.
After 2021, PROTAC entered a booming phase
.
Although no PROTAC drug has been approved to date, the technology opens a promising new chapter in drug discovery, opening up new treatment options
for some previously untreatable disease areas.
At present, PROTAC molecules have been widely explored by industry and academia, and its therapeutic fields have expanded from cancer to immune diseases, neurodegenerative diseases and viral infections ^[1].

Figure 1.
History of PROTAC ^[2].

The PROTAC molecule consists of three parts: one end is a ligand bound to the target protein, the other end is a ligand bound to E3 ligase, and the middle is connected
by the linker Linker.
Unlike traditional small molecule drugs that block the function of the target protein by inhibition, PROTAC induces target protein degradation
through the ubiquitin-proteasome system (UPS) by specifically binding to the target protein and E3 ligase, bringing the target protein closer to the E3 ligase.
The process of action of PROTAC is known as the "chemical knockout" pathway
.

PROTAC faces many DMPK research challenges mainly because PROTAC is a class of small molecule compounds
that violate the Rule of Five (also known as the Lipinski rule).
The Class of Drugs 5 principle is a set of guidelines for screening compounds for "druggability", that is, when the compound structure has more than 5 hydrogen bond donors, more than 10 hydrogen bond acceptors, molecular weight greater than 500, log P greater than 5, or rotatable bond greater than 10, the compound is likely to have poor absorption or permeability
.
Due to its structural characteristics, PROTAC generally belongs to the chemical space
outside the class drug 5 principle.
The high molecular weight of PROTAC (typically between 700-1200 Da), poor solubility, and low permeability pose challenges
to improve oral bioavailability in vivo.
To do this, developers must optimize the physicochemical and pharmacokinetic properties
of PROTACs.

It is administered with meals

Solubility is one of the determinants of the oral availability of
drugs.
PROTAC itself has poor water solubility and strong lipophilicity, which is not conducive to intestinal absorption
.
Studies have found that the solubility of PROTAC in simulated intestinal fluids, especially postprandial simulated intestinal ^{fluids, is greatly improved [3].}

This result suggests that PROTAC compounds administered after eating may result in better exposure to drugs in vivo
.
The clinical trial design of ARV-110 and ARV-471 also revealed that the phase I clinical administration pattern of these two PROTAC molecules is "once daily with meals"
.

Figure 2.
The solubility of PROTAC in different ^{buffers [3].}

Figure 3.
Clinical administration of ARV-110 and ARV-471

2

Optimize the Linker structure to improve PROTAC permeability

Permeability is another determining factor
affecting oral bioavailability.
Oral absorption of the drug requires passage through the membrane barrier of the epithelial cells of the small intestine; To degrade intracellular proteins, PROTAC also needs to enter target cells
.
Studies have shown that replacing PEG Linker with a 1,4-disubstituted benzene ring can significantly improve the cellular permeability of PROTAC[4^].

3

Optimize the Linker structure to improve PROTAC metabolic stability

After the compound is absorbed through the intestine, it is first metabolized through the liver or intestine before reaching the systemic circulation, which is called first-pass metabolism, which limits the oral absorption
of the drug.
To improve the oral bioavailability of PROTAC, improving metabolic stability to reduce first-pass metabolism is also an effective method
.
Various strategies such as changing the length and anchor of Linker, using a cyclic Linker, or changing the attachment site of Linker have been studied to improve the metabolic stability of PROTAC ^[5].

Figure 4.
Optimize Linker to improve PROTAC metabolic stability^[5].

4

Choose smaller E3 ligands

The properties of PROTAC are closely related
to the type of E3 ligand.
It has been reported that the E3 ligase ligands used in PROTAC are mainly CRBN, VHL, IAP and MDM2, of which CRBN and VHL are used most
frequently.
As can be seen from the figure below, CRBNs are smaller
compared to VHL ligands.
PROTAC containing VHL ligands is generally poorly absorbed orally due to its larger molecular weight ^[6].

PROTAC containing CRBN ligands has a smaller molecular weight and is more similar to "oral drugs"
.
The two PROTAC molecules ARV-110 and ARV-471 that entered clinical phase II are both CRBN E3 ligands
.
The search for new E3 ligands with smaller molecular weights is well worth exploring
.

Figure 5.
Common CRBN ligand and VHL ligand structures

5

Introduction of intramolecular hydrogen bonds

PROTAC typically has high polarity and multiple rotatable bonds, structures that are often difficult to permeate the lipid bilayer
of the cell membrane.
Recent studies have found that intramolecular hydrogen bond formation can improve cellular permeability
of PROTAC by reducing its polar molecular surface area.
Under the action of intramolecular hydrogen bonding, the PROTAC molecule changes from an elongated bar to a folded conformation ^[7], making it easier to penetrate the lipid bilayer
of the cell membrane.
For details, please refer to the public account article: PROTAC druggability research: solubility and permeability research strategy analysis
.

6

Apply prodrug strategies

Prodrug design is a common method
to improve the oral bioavailability of drugs.
Prodrugs
are obtained by structural modification of pharmacologically active compounds.
The prodrug itself has little or no activity, and the pharmacologically active parent drug
is released in vivo through enzyme catalysis.
One potential problem with designing prodrugs for PROTAC molecules is that it may further increase the molecular weight
of PROTAC.
Chemists engineered prodrugs
by adding lipophilic groups to the CRBN ligand of a PROTAC molecule.
The results showed that this prodrug design resulted in a significant increase in the bioavailability of PROTAC ^[8].

This is a prodrug based on a CRBN ligand design that can also be used for other PROTACs
with similar E3 ligands.

Figure 6.
Prodrug designs improve oral bioavailability of PROTAC[^8].

7

Molecular glue

PROTAC consists of three parts: two independent ligands and a Linker
.
Such heterogeneous bifunctional molecules give them a large molecular weight
.
Chemists are constantly exploring new strategies to reduce molecular weight and make PROTAC more "drug-like"
.
Molecular glues are considered to be a more "dense molecule" than PROTAC, and they can also trigger the formation
of ternary complexes like PROTAC.
For these reasons, molecular gums have better drug-like properties than PROTAC ^[9].

Figure 7.
Comparison of mechanisms of action of molecular glues and PROTAC^[9].

summary

The development of PROTAC technologies has opened up unprecedented therapeutic options for drug discovery, the most exciting of which is their potential
to target "undruggable" targets.
Despite the industry's optimism about some late-stage clinical studies, developers still face the challenge of optimizing the
pharmacokinetic behavior of PROTAC without compromising efficacy.
Strategies
to increase oral bioavailability of PROTAC are summarized in this article.

Through structural modification of PROTAC molecules, such as modifying Linker, introducing intramolecular hydrogen bonding, prodrug design; As well as choosing the appropriate mode of administration, such as with meals, can improve the oral bioavailability of PROTAC molecules
.
It is expected that drug developers from academia and industry will cooperate to solve the challenges of PROTAC in pharmacokinetics as soon as possible and release the huge potential
of PROTAC drugs.

References:

[1] Montrose K , Krissansen G W .
Design of a PROTAC that antagonizes and destroys the cancer-forming X-protein of the hepatitis B virus[J].
Biochem Biophys Res Commun, 2014, 453(4):735-740.

[2] Békés M, Langley DR, Crews CM.
PROTAC targeted protein degraders: the past is prologue[J].
Nat Rev Drug Discov.
2022 Mar; 21(3):181-200.

[3] Pike A , Williamson B , Harlfinger S , et al.
Optimising proteolysis-targeting chimeras (PROTACs) for oral drug delivery: a drug metabolism and pharmacokinetics perspective - ScienceDirect[J].
Drug Discovery Today, 2020, 25( 10):1793-1800.

[4] Farnaby W , Koegl M , Roy M J , et al.
Publisher Correction: BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design[J].
Nature Chemical Biology.

[5] Goracci L , Desantis J , Valeri A , et al.
Understanding the Metabolism of Proteolysis Targeting Chimeras (PROTACs): The Next Step toward Pharmaceutical Applications[J].
Journal of Medicinal Chemistry, 2020, 63(20):11615–11638.

[6] Poongavanam V, Kihlberg J.
PROTAC cell permeability and oral bioavailability: a journey into uncharted territory[J].
Future Med Chem.
2022 Jan; 14(3):123-126.

[7] Atilaw Y , Poongavanam V , Nilsson C S , et al.
Solution Conformations Shed Light on PROTAC Cell Permeability[J].
ACS Medicinal Chemistry Letters, 2020.

[8] Wei M , Zhao R , Cao Y , et al.
First orally bioavailable prodrug of proteolysis targeting chimera (PROTAC) degrades cyclin-dependent kinases 2/4/6 in vivo[J].
European Journal of Medicinal Chemistry, 2020, 209:112903.

[9] Maneiro M , Vita E D , Conole D , et al.
PROTACs, molecular glues and bifunctionals from bench to bedside: Unlocking the clinical potential of catalytic drugs[J].
Progress in Medicinal Chemistry, 2021.

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