Covalent drugs refer to those drug molecules that form strong bonds with receptors through the formation of covalent bonds. It all began with aspirin in 1899, gained prominence with ibrutinib in 2013, and reached great recognition in the anti-SARS-CoV-19 drug Paxlovid (Nirmatrelvir).
While most covalent drugs on the market come from the small molecule camp, the unique physicochemical properties of peptides and their inherent advantages over small molecules have led to increasing attention in the development of peptide covalent drugs. The active component nirmatrelvir (Figure 1) in the anti-SARS-CoV-2 drug Paxlovid is indeed a peptidomimetic covalent drug.
Protein-Protein Interactions
Compared to small molecules, peptide drugs inherently possess natural advantages in terms of selectivity and off-target effects, especially when interfering with biological processes based on protein-protein interactions (PPI) as the disease mechanism. These interactions regulate normal cellular functions and undertake new biological functions.
Understanding the physical contacts between proteins within cells is crucial for comprehending cellular physiology under both normal and disease conditions. Many diseases, including cancer, result from disrupted regular PPI and the overtake of abnormal PPI, including those from endogenous cellular proteins and pathogenic proteins. Hence, restoring normal PPI or inhibiting abnormal PPI holds significant clinical implications, which align with the strengths of peptide drugs.
Peptide Covalent Inhibitors
While some small molecule ligands can impact PPI and some have been approved for clinical use, the relatively flat topology and large interface of PPI, as well as the absence of traditional binding pockets or catalytic sites, pose challenges for small molecule interference. In such scenarios, the development of peptide and peptidomimetic PPI inhibitors becomes a logical pathway.
The binding sites of these peptides are determined by their secondary or tertiary structures in their bound state, often having larger binding interfaces compared to small molecules. Researchers have found that stabilizing biologically active secondary or tertiary structures through intramolecular cross-linking and other modifications can enhance ligand target affinity, cellular uptake, and stability, highlighting another advantage of peptide drug development.
Despite their inherent size advantage, relying solely on traditional non-covalent binding mediated by peptides is often insufficient for effectively intervening in target PPI, especially for protein receptors considered undruggable. In this dilemma, the concept of covalent drugs provides a fresh strategy for developing peptide inhibitors. The combination of peptide inhibitors with covalent drugs has given rise to the field of peptide PPI covalent inhibitors.
Covalent inhibition offers several advantages: introducing covalent binding regions can enhance the performance of moderately affine binders, significantly prolonging their target residence time and duration of action. This minimizes unnecessary scaffold structures, thereby enhancing their drug-like properties. Furthermore, irreversible inhibition can generate potent effects that remain competitive even in the presence of high concentrations of endogenous ligands, effectively reducing dosages, optimizing pharmacokinetics, and enhancing drug safety.
In recent years, drugs with covalent binding modes have achieved success in clinical settings, reigniting interest in irreversible inhibitors. Particularly, incorporating active groups (electrophilic groups, referred to as warheads) into peptides has become a focal point in developing peptidomimetic covalent inhibitors that leverage the impact of non-covalent interactions. It’s noteworthy that such inhibitors often target non-catalytic nucleophilic residues near binding sites, expanding their scope beyond enzyme inhibition. An increasing number of peptide-targeting covalent inhibitors are entering clinical research, mainly targeting cysteine or lysine residues on receptors.
Types of Peptide Covalent Inhibitors
- Acrylamide
Among the covalent inhibitors available on the market, the most common electrophilic group used as a warhead is acrylamide. Designers utilize nucleophilic groups on receptor proteins, such as the thiol group of cysteine side chains or the amino group of lysine side chains, to achieve irreversible covalent reactions with acrylamide through Michael-addition reactions.
Acrylamide is a commonly used electrophilic reagent for protein targeting and has been employed in designing peptide-based covalent inhibitors of the E3 ubiquitin ligase SIAH. SIAH impacts HIF-1α transcription factor levels and regulates critical cell activities in cancer development.
Researchers have converted suboptimal non-covalent inhibitors into upgraded covalent inhibitors BI-107F9 and BI-107G3 by incorporating acrylamide warheads through amino modifications of lysine and ornithine side chains. Cell-penetrating peptide (CPP) sequences TAT and P10 were introduced at the N-terminus of these two peptides.
The resulting peptide covalent inhibitors can target cysteine residues (SIAH Cys130) near the binding site. Ideal inhibition is mediated by the Michael-addition reaction between the thiol group of the cysteine side chain and the acrylamide warhead on the peptide molecule.
- Chloroacetamide
Chloroacetamide is another electrophilic group that can covalently interact with cysteine residues on receptors. It has been used to generate covalent antagonists of BFL-1. The resulting peptide 138C7 covalently binds to the Cys55 residue of the BFL-1 receptor. Inhibitor analogs similar to 138C7, namely 138C5 and 138C8, effectively induce apoptosis in SKMEL28 melanoma cell lines with high BFL-1 expression.
- Vinyl Sulfonamide
Vinyl sulfonamide serves as an electrophilic group for covalent inhibitors targeting the Ras (rat sarcoma) protein. This strategy is applicable to specific oncogenic mutations in Ras (G12C). In the development of Ras covalent inhibitors, vinyl sulfonamide was identified as a promising electrophilic reagent with sufficient reactivity and selectivity. The initial designed covalent inhibitor α3βHBSSOS-6 exhibited activity against H358 lung cancer cells dependent on Ras G12C.
- Sulfonium
Sulfonium can also serve as electrophilic groups in peptide covalent inhibitors. For instance, the peptide B4-MC-I can selectively modify the Cys55 residue of the BFL-1 receptor and induce apoptosis in cell lines expressing BFL-1.
- Isothiocyanate
Isothiocyanate also serves as an electrophilic group for modifying peptide covalent inhibitors. One example uses isothiocyanate to target Lys574 on CD4.
- Thioester
Thioesters also possess some electrophilicity and can be used as warhead structures for peptide covalent inhibitors, although their reactivity is relatively weaker.
- Aryl Sulfonyl Fluoride
In various forms of cancer, overexpressed cancer proteins Mdm2 and Mdm4 prevent p53 transcriptional activity by ubiquitination and subsequent proteasomal degradation. Thus, inhibiting this interaction can restore endogenous p53 activity and reduce tumor growth. Peptide covalent inhibitors containing aryl sulfonyl fluoride electrophilic groups were developed to target lysine and histidine residues in Mdm2 and Mdm4 receptors.
- Boronic Acid
Boronic acid mimetic dipeptides, represented by Bortezomib, are functionally classified as protease inhibitors. Bortezomib targets the 26S protease for the treatment of multiple myeloma and condylomatous lymphoma. Peptide covalent inhibitors containing boronic acid structure that reacts with the hydroxyl group of the serine side chain in a reversible covalent binding interaction.
Summary
While many small molecule covalent inhibitors have successfully entered the market, efforts to target PPI interfaces with small molecules still face significant challenges. Peptides fill the gap left by small molecule covalent inhibitors in this field, and the development of reversible or irreversible covalent peptide inhibitors involves previously considered undruggable proteins, ranging from cancer-related signaling to viral receptors.
The use of peptide-based covalent inhibitors targeting specific receptor residues, mainly cysteine but also including lysine, histidine, and serine, is a growing field. However, the range of targetable amino acids needs to be expanded. Another limitation is cellular uptake of peptides, which can be overcome by reducing inhibitor molecular weight and adding extra cell-penetrating peptide sequences. More in vivo studies are needed to explore the applicability of peptide covalent inhibitors, including the selection of electrophilic groups (warheads). Covalent peptide inhibitors will combine enhanced protein surface recognition capabilities with reversible or irreversible covalent binding modes. They have proven to be effective in targeting previously considered undruggable protein receptors.
References:
- Pelay-Gimeno, M. et al., Structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes, Angew Chem Int Ed., 2015, 54, 8896-8927.
- Yoo, D. Y. et al., Covalent targeting of Ras G12C by rationally designed peptidomimetics. ACS Chem Biol., 2020, 15, 1604-1612.
- Liu, N. et al., Selective covalent targeting of anti-apoptotic BFL-1 by a sulfonium-tethered peptide, Chembiochem., 2021, 22, 340-344.
- Baek, S. et al., Structure of the stapled P53 peptide bound to Mdm2, J Am Chem Soc., 2012, 134, 103-106.
- Chen, D. et al., Bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives, Curr Cancer Drug Targets., 2011, 11(3), 239-253.
- Paulussen, F. M. et al., Peptide-based covalent inhibitors of protein-protein interactions, J Pept Sci., 2023, 29, e3457.