Covalent Inhibitors Targeting Undruggable Protein

With the rise of genomics and proteomics, many clinically significant targets have been discovered in human diseases. However, a relatively small proportion of traditional druggable targets exists, and many targets are categorized as undruggable due to their flat functional surfaces and a lack of well-defined pockets for ligand interactions, posing significant challenges in drug design.

Undruggable Proteins

1. Small GTPase RAS family proteins, including KRAS, HRAS, and NRAS, belong to the small GTPase category. These proteins are considered undruggable due to the lack of targetable pockets on their surfaces.
2. Transcription factors (TFs) are associated with various human diseases and participate in numerous biological processes. Most TFs cannot be targeted by conventional small molecules due to their structural heterogeneity and lack of accessible binding sites. Current research primarily focuses on targeting p53 and Myc.
3. Protein-protein interaction (PPI) and their networks play a significant role in biological processes and cell cycle regulation, providing another potential pathway for treating complex diseases. RAS and TFs such as p53 and Myc are also influenced by PPI networks. Some PPIs with flat interaction surfaces are more challenging to target than others, making them undruggable to a certain extent. Classical PPI-related proteins include the anti-apoptotic members of the Bcl-2 family. Additionally, intrinsically disordered proteins with highly dynamic structures, lacking binding cavities, interacting with various protein partners, are also considered undruggable.
4. Epigenetic targets play a crucial role in regulating gene expression patterns and impact various biological processes and diseases. Epigenetic modifications include DNA methylation, histone modifications, non-coding RNA, chromatin remodeling, and other epigenetic enzymes. Targeting these epigenetic targets may reveal potential mechanisms for various diseases.
5. Phosphatases are divided into two types based on structural features: protein tyrosine phosphatases (PTPs) and protein serine/threonine phosphatases (PSTPs). Due to the structural similarities of each phosphatase, there are issues of low selectivity and unavoidable side effects.

Covalent Modulation

Covalent inhibitors, also known as irreversible inhibitors, are a class of inhibitors that form a covalent bond with amino acid residues of the target protein through mildly reactive functional groups, providing additional affinity. Covalently bound targets can be continuously inhibited until protein degradation and regeneration. Compared to non-covalent inhibitors, covalent inhibitors have the advantages of sustained inhibition and longer residence time. Covalent inhibitors can also reduce dosage, improve compliance, and avoid potential resistance mechanisms. For example, Nirmatrelvir in Paxlovid, approved for COVID-19, is an inhibitor of the main protease (Mpro) of SARS-CoV-2, highlighting the importance of cysteine-reactive covalent functional groups in targeting the active site of proteases. Moreover, covalent inhibitors can target undruggable proteins lacking surface pockets, potentially expanding the scope of treatment. The approval of the KRAS inhibitor Sotorasib is a milestone in the development of covalent drugs.

KRAS Covalent Inhibitors

KRAS plays a crucial role in signaling pathways involved in cell growth and survival and is the most predominant mutated subtype in the RAS family, particularly associated with pancreatic, colorectal, and lung cancers. KRAS proteins transition between inactive and active states, with GDP-bound KRAS in the inactive state and GTP-bound KRAS in the active state. This transition is regulated by two factors: (1) guanine nucleotide exchange factors (GEFs), such as the SOS protein, catalyzing the transition between KRAS and GTP-binding states, and (2) GTPase-activating proteins (GAPs), promoting the hydrolysis of GTP bound to KRAS, inhibiting KRAS activity.

Directly targeting KRAS poses many challenges. KRAS shares high homology with NRAS and HRAS, and insufficient specificity of KRAS inhibitors may inhibit the activities of NRAS and HRAS, leading to potential side effects. The known active functional domains (potential binding sites for drugs) in KRAS are mainly the pocket-like domains where KRAS binds to GDP and GTP.

Common mutation sites in KRAS include codons 12, 13, and 61, with codon 12 being the most frequent mutation site. In the KRAS^G12C mutant, small molecules covalently bound to the cysteine on the mutant protein are more likely to bind to the GDP-binding KRAS protein. This binding reduces the affinity between GTP and KRAS, preventing the replacement of GDP with GTP, thereby locking the KRAS^G12C mutant in an inactive state. The discovery of this binding pocket on the KRAS^G12C mutant has accelerated the development of several small molecule covalent inhibitors specifically targeting this mutant. Among them, Sotorasib and Adagrasib are already in clinical use, and over a dozen are undergoing clinical trials.

EGFR Covalent Inhibitors

Epidermal Growth Factor Receptor (EGFR) is a member of the tyrosine kinase receptor family, a typical transmembrane receptor that initiates signal cascades during ligand-induced dimerization, activating its tyrosine kinase and various downstream effectors. Additionally, it is involved in embryonic development, stem cell division, and is associated with cell proliferation, mitosis, and the development of various cancers such as non-small cell lung cancer, breast cancer, glioblastoma, head and neck cancer, cervical cancer, bladder cancer, etc. Therefore, EGFR has become a promising target for designing and developing anticancer drugs.

Targeted drugs for EGFR are tyrosine kinase inhibitors (TKIs), inhibiting the kinases in the cytoplasm to prevent activation of the EGFR signaling pathway. First-generation TKIs, such as gefitinib and erlotinib, selectively bind to the ATP binding site of EGFR through non-covalent bonds, inhibiting EGFR phosphorylation and significantly slowing down the progression of targeted treatment in non-small cell lung cancer (NSCLC). However, resistance has gradually emerged: only 10-19% of advanced NSCLC patients respond to gefitinib. Subsequent research indicates that the reduced sensitivity of NSCLC patients to gefitinib or erlotinib is associated with EGFR-specific activating mutations.

Mutations in EGFR limit the effectiveness of reversible EGFR TKIs and render them undruggable. To overcome this issue, irreversible EGFR TKIs, namely second-generation EGFR TKIs, are designed to covalently bind to the binding site, enhancing persistent inhibition of tumor cells. Compared to first-generation EGFR TKIs, second-generation EGFR TKIs, such as afatinib, dacomitinib, and neratinib, have acrylamide as michael receptor that irreversibly bind to Cys797 at the ATP binding site, demonstrating stronger inhibitory effects in clinical practice. However, second-generation EGFR inhibitors are still not suitable for treating patients with resistance mutations after using first-generation EGFR inhibitors, and they may also lead to resistance, often associated with the T790M mutation. Therefore, third-generation EGFR TKIs like WZ 4002, osimertinib, and rociletinib, specifically designed for the T790M mutant, have been developed. These third-generation drugs retain the acrylamide group, covalently binding to Cys797, but replace the quinazoline part of the first and second-generation compounds with pyrimidine, increasing selectivity for T790M, with higher affinity than WT-EGFR. Thus, the development of covalent EGFR inhibitors is highly attractive.

p53 Covalent Modulators

Studies indicate that approximately half of human cancers, including serous ovarian cancer, squamous cell lung cancer, small cell lung cancer, triple-negative breast cancer, and squamous esophageal cancer, exhibit alterations in the p53 gene, leading to loss of p53 function or reduced p53 expression. As a tumor suppressor closely associated with protein-protein interactions (PPIs), p53 plays a crucial role in regulating gene expression, promoting tumor cell cycle arrest, apoptosis, and DNA repair. It can activate nearby or distant genes in response to enhancers while indirectly inhibiting the transcription of many genes. P53 can be classified into mutant and wild-type forms, where mutant p53 promotes tumor development, while wild-type p53 has a broad-spectrum tumor suppressor effect. TP53 mutations typically decrease the expression of p53 protein or produce non-functional variants, impairing its anti-cancer characteristics. Therefore, therapeutic strategies are needed to restore p53 function. However, most small molecules aim to inhibit the activity of overexpressed proteins, making p53 a undruggable target.

• Directly targeting p53 with covalent modulators

In 2022, the team led by Kevan M. Shokat developed a small molecule covalent inhibitor, KG13, for the p53-Y220C mutant. This inhibitor is specifically designed to bind to the p53 Y220C mutant, restoring the thermal stability of the p53 protein to the level of wild-type p53 and activating the expression of downstream genes. Researchers designed 13 small molecules targeting the pocket structure formed by p53 Y220C. After a series of structural modifications and screenings, KG13 was selected as the optimal small molecule compound, showing the highest covalent labeling rate and thermal stability recovery. Furthermore, cells treated with KG13 showed p53 Y220C-dependent activation of p53 target genes, inhibition of cell growth, and increased caspase activity.

• Covalent p53-MDM2 PPI inhibitors

MDM2 and MDMX are negative regulators of p53, mediating its degradation in normal cells by directly binding to its N-terminus. This maintains p53 at low expression levels. The main mechanism of p53 degradation involves the ubiquitination mediated by the E3 ubiquitin ligase MDM2, leading to proteasomal degradation of p53. Since MDM2-mediated ubiquitination and degradation depend on its direct interaction with p53, researchers have been searching for small molecules that can inhibit this interaction to stabilize p53 and restore its activity. While most p53-MDM2 inhibitors are non-covalent, some small molecule inhibitors targeting p53-MDM2 have been found to be covalent.

Mcl-1 Covalent Inhibitors

Mcl-1 is an important anti-apoptotic member of the Bcl-2 protein family, playing a crucial role in the development of various human cancers. Targeting the BH3 binding groove of Mcl-1 is a promising approach to inhibit its function and has become a hot spot in anticancer drug development. In this regard, Lee et al. designed a drug design strategy based on variable structural sites near the BH3 region relative to the binding site. They used the covalent inhibitor MAIM1 to bind to Cys286, effectively inhibiting Mcl-1 activity. This compound tightly binds to Mcl-1, providing a potential new approach and precursor compound for anti-apoptotic tumor treatment.

References:

1. Lu, Y., et al., Emerging Pharmacotherapeutic Strategies to Overcome Undruggable Proteins in Cancer, International journal of biological sciences, 2023, 19(11), 3360-3382.
2. Xie, X., et al., Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials, Signal transduction and targeted therapy, 2023, 8(1), 335.
3. Guiley, K. Z. and Shokat, K. M., A Small Molecule Reacts with the p53 Somatic Mutant Y220C to Rescue Wild-type Thermal Stability, Cancer Discov., 2023, 13(1): 56-69.
4. Lee, S., et al., Allosteric Inhibition of Anti-Apoptotic MCL-1, Nat Struct Mol Biol., 2016, 23(6): 600-607.