Advances in covalent drug discovery

2022/10/12
M2 Zhai1

ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary

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ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary

3Introduction
Compounds that by design are intended to form a covalent bond with a specific molecular target.

Juswinder Singh, Journal of Medicinal Chemistry. 2022, 65, 5886

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Non-Covalent and Covalent Inhibitors
Non-covalent inhibitor
Non-covalent inhibitors bind to theirtargets in equilibrium and in a reversible
manner.

Covalent inhibitor
Covalent inhibitors bind to their targets in a twostep manner – the formation of initial noncovalent complex being reversible and formationof final covalent complex being irreversible.

Ruddraraju, K. V.; Zhang, Z. Mol. BioSyst. 2017, 13, 1257.

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Timeline of Covalent Inhibitor Drugs

Boike, L.; Henning, J.N. Nature Reviews. 2022, 13, 1257.

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Application of Covalent Inhibitor Drugs
FDA-approved covalent drugs (2011–2019)

Approved covalent drugs by therapeutic indication.
Sutanto, F.; Konstantinidou, M.; Dömling, A., RSC Medicinal Chemistry. 2020, 11, 876.

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The advantages and disadvantages
Advantages
Ø Improving efficiency.
Ø Lowering the dose.
Ø Increasing compliance due to lessfrequent dosing.
Ø Reducing the possibility of drugresistance.
Ø Targeting shallow binding sites.

Disadvantages
Ø May cause unexpected toxicity or
hypersensitivity.
Ø May cause drug-induced toxicity.
Ø May not be suitable for targets thatare rapidly turned over/ degraded byenzymes.
Ø May cause problems in choosing thecorrect warhead targeting.

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ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary9Different Warheads of TCIs
The distribution of reaction mechanisms from the CovPDB database

The distribution of the FDA approved drugs according to their targets
and labeled residues.

Péczka,N.; Orgován, Z.; et al, Expert Opinion on Drug Discovery, 2022, 17:4, 413

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ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary11

Classification of Covalent Inhibitors

Tuley, A.; Fast, W. Biochemistry. 2018, 57, 3326.
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Covalent Reversible Inhibitorsl Reversiblel Selectivel Ki* describes the overall dissociation constant of the two steps

The low pKa of the α-proton makes the reaction reversible.
Tuley, A.; Fast, W. Biochemistry. 2018, 57, 3326.

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Slow Substratesl Reversiblel Inhibitor recognized as substrate for the enzymel Covalent intermediate further decomposes into free enzyme and non-active product(P）

Slow hydrolysis of the pseudo thiourea through the normal catalytic mechanism
leads to release of O-ureido and recovered active enzyme.
Tuley, A.; Fast, W. Biochemistry. 2018, 57, 3326.

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Residue-Specific Reagentsl Irreversiblel The least selectivel Used only in vitro as biochemical toolsl Influenced by chemo selectivity for particular nucleophiles instead of noncovalentaffinityHigh concentration leads to nonspecific enzyme inhibition, illustrating thenonselective nature of residue-specific reagents.
Tuley, A.; Fast, W. Biochemistry. 2018, 57, 3326.

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Affinity Labelsl Irreversiblel Site selective inhibitionl Moiety with non-covalent binding affinity + reactive group (typically a poorelectrophile)l Dissociation from covalent complex E–I to non-covalent complex E•I can be ignored

The effective molarity of
the reactive group near thesite of enzyme
modification is raised by
the non-covalent binding.

Tuley, A.; Fast, W. Biochemistry. 2018, 57, 3326.

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Mechanism-Based Enzyme Inactivatorsl Irreversiblel Selectively Bind to active site of enzymes
l Processed by catalytic mechanism to give reactive species

Tuley, A.; Fast, W. Biochemistry. 2018, 57, 3326.

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ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary

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Kinase-targeting Covalent Inhibitors
Kinases play crucial roles in regulating various cellular activities by catalyzing the phosphorylation of biomacromolecules.

Covalent inhibitors use electrophilic warhead groups to react with nucleophilic kinase residues such as cysteine to formcovalent bonds to inhibits ATP binding.

Goebel, G.; Wu, P. Trends Pharmacol. Sci. 2022, 43, 10.

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Kinase-targeting Covalent Inhibitors
Bruton's tyrosine kinase (BTK)BTK plays a crucial role in B cell development as it is required for transmitting signals from the pre-B cell receptor that
forms after successful immunoglobulin heavy chain rearrangement.

Arter, C.; Trask, L. etc. J. Biol. Chem. 2022, 298, 102247.

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Kinase-targeting Covalent Inhibitors
BTK-targeting covalent inhibitors block signaling through BTK inhibition by forming a covalent bond with Cys-481 in theATP binding domain of BTK.

Ibrutinib

Zanubrutinib

Acalabrutinib

Tirabrutinib

Approved drugs that treat cancers, including mantle cell lymphoma, chronic
lymphocytic leukemia, and Waldenström's macroglobulinemia.
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Joseph R et al. eLife, 2022, 9:e60470.

Kinase-targeting Covalent Inhibitors
Epidermal growth factor receptor (EGFR) is a transmembrane protein that is activated by binding of its specific ligands,
including epidermal growth factor and transforming growth factor α (TGFα).

Tamirat, MZ., et al. PLoS ONE, 2019, 14: e0222814.

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Kinase-targeting Covalent Inhibitors
EGFR-targeting covalent inhibitors
Targeting cys-797 in EGFR with covalent inhibitors is associatedwith the treatment of a wide variety of tumors.

Approved EGFR covalent inhibitors

T790M EGFR covalent inhibitors
Afatinib covalently binds to cysteine 797 of EGFR via
a Michael addition (IC50 = 0.5 nM)
Abourehab, M. et al. Molecules, 2021, 26, 6677

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Kinase-targeting Covalent Inhibitors
PROTACs are bifunctional molecules with a ligand at one end binding to a target of interest and an E3 ubiquitin ligase
binder at the other end to recruit E3–E2 ligases for ubiquitin- mediated proteasomal degradation.

Covalent kinase inhibitors were further developed as kinase-targeting PROTACs.
••Schiemer, J. et al. Nat Chem Biol, 2021, 17, 152–160.

BC5P was developed by linking a aminopyrazole based BTK inhibitor to a
known ligand of the E3 ligase cIAP via five polyethylene glycol (PEG)
molecules.
Treatment of THP-1 cells with BC5P led to a dose-dependent loss of BTK, witha half-maximum degradation concentration (DC50) of 182 ± 57 nM.

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ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary25

K-Ras-targeting covalent inhibitors
K-Ras and its biological roles
The K-Ras protein is a GTPase which converts
the nucleotide GTP into GDP.

KRAS Mutants are Prominent Oncogenic Drivers

KRAS was considered undruggable because of its relatively smooth surface as well as the high affinity of GTP to theGTP/GDP-binding pocket

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KRAS-targeting Covalent Inhibitors
Covalent KRAS(G12C) inhibitors
Compounds that covalently and irreversibly
bound to the cysteine residue of the KRASG12C.

An allosteric pocket beneath the switch II region near the mutant cysteine was discovered

Compounds block K-Ras(G12C) interactions, decrease viability and increase apoptosis of
G12C-containing lung cancer cell lines.

Ostrem, JM. et al. Nature, 2013, 503, 548–551.

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KRAS-targeting Covalent Inhibitors
Covalent KRAS(G12C) inhibitors
Sotorasib (AMG510) obtained FDA approval in 2021 tobecome the first therapy to directly target the KRAS
oncoprotein in tumors

Adagrasib (MRTX849) and other direct KRASG12C inhibitors are currently
being investigated in multiple clinical trials.

Preclinical compounds

Canon, J. et al. Nature, 2019, 575, 217–223.

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KRAS-targeting Covalent Inhibitors

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ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary

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Lysine-Targeting Reversible Covalent Inhibitorsl Reversible covalent inhibitorsl Low Koff to maximize the benefit of the warhead, and also
to achieve long-lasting inhibition.
l Diazaborines and related B−N heterocycles have also been
explored as enzyme inhibitors and as reversible linkers for
drug delivery to cancer cells

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Lysine-Targeting Reversible Covalent Inhibitorsl A new warhead RMR1

Reja, Rahi.m. et al, J. Am. Chem. Soc. 2022, 144, 1152

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Lysine-Targeting Reversible Covalent Inhibitors

Replacing the APBA warhead with RMR1 (P5) gave
even greater potency with an IC50of 1.3 μM.
The reversibility of the diazaborine conjugation
was confirmed

Reja, Rahi.m. et al, J. Am. Chem. Soc. 2022, 144, 1152

• A novel lysine conjugation chemistry• RMR1 can be grafted on to a peptide scaffold to create
potent reversible covalent inhibitors.

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ContentsØIntroductionØDifferent warheads of TCIs
ØClassification of covalent inhibitorsØDevelopment of covalent inhibitors- Kinase-targeting covalent inhibitors- K-RAS-targeting covalent inhibitors- Lysine-targeting reversible covalent inhibitorsØConclusion and summary

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Conclusions• Expanded covalent warhead toolbox allows for selective targeting of specificamino acid residues.
• The approvals of successful drugs showcase the evolution of covalent drug
discovery from a serendipitous effort to a field with established roadmaps forsuccess.
• Potential in new modalities such as PROTACs.

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