Biswajit Biswas, Yen-Hua Huang, David J. Craik * and Conan K. Wang *Human kinases are recognized as one of the most important drug targets associated with cancer. There are >80 FDA-approved kinase inhibitors to date, most of which work by inhibiting ATP binding to the kinase. However, the frequent development of single-point mutations within the kinase domain has made overcoming drug resistance a major challenge in drug discovery today. Targeting the substrate site of kinases can o ff er a more selective and resistance-resilient solution compared to ATP inhibition but has traditionally been challenging. However, emerging technologies for the discovery of drug leads using recombinant display and stabilization of lead compounds have increased interest in targeting the substrate site of kinases. This review discusses recent advances in the substrate-based inhibition of protein kinases and the potential of such approaches for overcoming the emergence of resistance.
Protein kinases catalyze one of the most fundamental biochemical reactions of life. They transfer the g-phosphate of a purine nucleotide triphosphate (ATP/GTP) to the hydroxyl group of their substrate proteins. 1 Over 500 human kinases carry out this type of reaction to regulate key cellular processes that range from cell growth to cell cycle progression and cell di ff erentiation, proliferation, metabolism, and apoptosis. However, when kinases become dysregulated, they transition into drivers of disease, commonly cancer. 2,3 The rst cellular proto-oncogene, identi ed over 40 years ago, was found to encode c-Src kinase. 4 Kinases are amongst the most common cancer gene-encoded protein domains and are attractive drug targets. 5
Inhibiting the ATP binding site of kinases was initially viewed as an unsurmountable challenge because of the high intracellular ATP concentration. 6 However, with the FDA approval in 2001 and the clinical success of BCR-Abl inhibitor imatinib, interest in developing oral ATP-competitive kinase inhibitors skyrocketed. 7,8 As of April 2024, 81 protein kinase inhibitors have been approved by the FDA, most of which work by inhibiting ATP binding to the kinase domain. 9 Moreover, six additional inhibitors have been approved for lipid kinases and a staggering 600 kinase-targeting agents are under clinical trials according to a report in 2021. 6 The major challenge in drug discovery today, however, is the rapid emergence of drug resistance, typically developing from such acquired mutations within the kinase domain that prevent ATP-inhibitor binding. 8,10 Another, now pressing challenge, relates to poly-pharmacology, i.e. , ATP-competitive inhibitors can act on more than one kinase. Interestingly, targeting multiple kinases can have favourable e ffi cacy e ff ects but also lead to adverse patient outcomes in other cases, 11,12 pointing to the need in general of being able to ne-tune inhibitor activity. To enable greater selectivity and combat drug resistance, there are increasing calls to explore non-ATP-mediated kinase inhibition. 6,13 Substrate binding is another molecular interac-tion essential to kinase function. Unlike the ATP-binding site, the substrate-binding site of protein kinases is less conserved and thus off ers better selectivity. 14 Should mutations be acquired within the site to reduce inhibitor binding, the coin-cident e ff ect would be reduced kinase activity. However, the substrate binding site has a shallow and open surface, which has made the design of small molecule inhibitors di ffi cult. 14,15 Furthermore, the molecular details of kinase –substrate inter-actions have been sparse until recently, which has further hindered the development of substrate-site inhibitors. 15 Despite these di ffi culties, the eld has progressed, and a multitude of kinase substrate-site inhibitors have been reported. In this review, we aim to provide an up-to-date discussion of substrate-site inhibitors and their potential in overcoming drug resistance. As background for the need for such inhibitors, we summarize the FDA-approved ATP-competitive kinase inhibi-tors and highlight mutations to the ATP-binding region asso-ciated with their drug resistance. For comprehensive discussions of ATP-competitive inhibitors, we refer readers to several recent reports. 6,8,9,12 Substrate-site inhibitors, on the other hand, have been reviewed in very few articles, each focusing on speci c modalities without providing a complete overview and not including recent studies. 14,16,17 This article will cover both small molecule and peptide-based substrate-site inhibitors, but with a focus on the latter. This focus on peptides and their design technologies is because most re-ported substrate-site inhibitors have been peptides. Peptides naturally mimic the substrates of protein kinases and therefore present as promising leads in drug development. Finally, we discuss the evidence that explores the potential of substrate-site inhibitors in overcoming the emergence of drug resistance.
Human protein kinases can be classi ed based on their substrate speci city and/or sequence similarity. According to the amino acid they phosphorylate, most are named either serine/threonine kinases (STKs) or tyrosine kinases (TKs), with STKs (>300 reported) being more prevalent than TKs (>50). 18 Sequence analyses of these kinases have borne a separate and more granular classi cation scheme that begins with their division into eukaryotic (ePKs, 478 kinases) and atypical protein kinases (aPKs, 40 kinases), with the former, but not latter, having the ‘kinase catalytic domain ’.19 The ePKs are further divided into 9 groups, and these are, in order of abundance: TK (tyrosine kinase), CAMK (Ca 2+ /calmodulin-dependent kin-ase),TKL (tyrosine kinase-like), AGC (protein kinase A, G and C related), CMGC (Cdk, GSK, MAPK, Cdk-like related), STE (STE20, STE11, and STE7 related), CK1 (casein kinase 1), RGC (receptor guanylyl cyclase), and “others ”.20,21 PKA (c-AMP dependent protein kinase) is a prototypical example of both an STK and an AGC, and its crystal structure was the rst to reveal the bi-lobal fold of the kinase catalytic domain. 22 Another example, Abl, is prototypical of TKs, and its kinase domain was the rst to be successfully drugged for the treatment of cancer. 8
Fig. 1a shows a typical catalytic cycle carried out by the protein kinase domain once activated, usually by being itself phosphorylated. The ATP binding, magnesium complexation and substrate recognition and positioning steps at the catalytic site are followed by phosphoryl transfer, and nally release of the substrate (phosphorylated) and ADP products. 23 In some cases the order of these steps varies, for instance substrate binding can precede ATP binding, and ADP can be released before substrate dissociation. 24 Regardless, when the catalytic cycle becomes dysregulated, it results in aberrant phosphory-lation and disease. 25 As an example, a constitutive active mutant of Abl is the oncogenic cause of chronic myeloid leukaemia. 26 Kinase binding to either ATP or substrates is governed by di ff erent binding properties. Generally, ATP binding is driven by moderate a ffi nity in the 10 –100 mM range combined with high intracellular ATP concentrations of 1–10 mM. 27 By contrast, the substrate binding a ffi nity is substrate and kinase-dependent and can involve regions outside the substrate sequence motif (a contiguous ∼10 amino acid region around the acceptor residue) and catalytic site. For example, an N-terminal SH2 domain o en aids the positioning of protein substrates for catalysis by Src TKs. 28 Abl utilizes the SH2 and SH3 domains adjacent to its kinase domain to mediate substrate recognition. 29 Substrate binding is thus likely stronger than the mM a ffi nity for a peptide representing the sequence motif. 30 It is also di ffi cult to generalise for the substrate concentrations inside cells. While there are ∼700 000 potential intracellular phosphorylatable sites, kinases vary greatly in the number of sites they phosphorylate and their substrate recog-nition motifs. 24
The ePK catalytic domain has approximately 250 amino acid residues and contains the essential structural features for cat-alysing substrate phosphorylation (Fig. 1b). The domain has an N-terminal and a C-terminal lobe connected through a hinge region. 31 The N-terminal lobe is made up of ve b-strands ( b1-b5) and one a-helix (aC-helix), while the C-terminal lobe comprises four short b-strands ( b6-b9) and seven a-helices ( aD-aI). 32 The C-terminal lobe also contains a exible polypeptide segment, which is divided into the catalytic, activation and ‘P+1 ’ loops, and is important for catalysis and coordinating kinase binding to magnesium, ATP and substrates. 32 The conformation of the activation loop can change between activated and inac-tivated kinase states to facilitate or inhibit/block binding of ATP and substrates. 33
The ATP-binding site is a deep pocket formed between the two lobes of the kinase domain. 15 Once bound, ATP resides near 23 residues in PKA (PDB ID: 3X2V) and 17 residues in Abl (PDB ID: 2 G2I, residues within 5 Å proximity). In general, the adenine of ATP is surrounded by conserved hydrophobic residues and forms hydrogen bonds to the hinge region. 34 The remainder of ATP binds to a hydrophilic channel that extends towards the substrate binding site, usually interacting with the N-terminal lobe through an AxK motif (Ala, x, Lys) and a glycine-rich loop (GxGxxG motif) that binds with the triphosphate group and the ribose moiety. 35,36 This phosphate binding region of the N-terminal lobe between b1 and b2 containing the AxK motif and glycine-rich loop is also known as the ‘P-loop ’.37,38 In the activated kinase state, the DFG motif (Asp, Phe, Gly) of the activation loop positions the ATP for phosphotransfer. 39 The majority of the approved kinase inhibitors target this ATP-binding site for inhibiting phosphorylation. Some inhibi-tors, as elaborated on later, engage adjacent regions outside the ATP pocket that are not occupied by ATP in attempts to increase inhibitor potency and/or selectivity. The ‘entrance ’ and ‘buried ’ regions are two such regions and have structural and sequence diversity among di ff erent kinases. 40 The entrance region resembles a solvent-exposed hydrophobic slot and access to it is controlled by the conformation of the DFG motif. 41 Access to the buried region is controlled by a single amino acid residue in the hinge region - known as the ‘gatekeeper residue ’.42 Mutation of the gatekeeper residue is a predominant cause of drug resis-tance for ATP competitive inhibitors. 43
The substrate-binding site is a shallow cle adjacent to the ATP-binding site in the C-terminal lobe. 15 The co-crystal struc-ture of PKA bound to a 20-amino acid peptide substrate, iden-ti ed 32 residues that constitute this site (PDB ID: 3X2V, within 5 Å proximity). Compared to those of STKs, the substrate-binding sites of TKs are deeper to accommodate the larger tyrosine acceptor residue. 24 Generally, peptide substrates bind the substrate-binding site in an extended conformation. 30 In this canonical binding mode, the substrate phosphorylation site is secured by the ‘P+1 ’ loop, which in turn is anchored to the aF-helix. The HRD-arginine of the catalytic loop anchors the primary phosphate. 36 Residues upstream of the phosphoryla-
