Human protein kinases are highly-sought-after drug targets, historically harnessed for treating cancer, cardiovascular disease, and an increasing number of autoimmune and inflammatory conditions. Most current treatments involve small molecule protein kinase inhibitors that interact orthosterically with the protein kinase ATP-binding pocket. As a result, these compounds are often poorly selective and highly toxic. Part I of this series reviews the role of PKC isoforms in various human diseases, featuring cancer and cardiovascular disease, as well as translational examples of PKC modulation applied to human health and disease. In the present Part II, we discuss alternative allosteric binding mechanisms for targeting PKC, as well as novel drug platforms, such as modified peptides. A major goal is to design protein kinase modulators with enhanced selectivity and improved pharmacological properties. To this end, we use molecular docking analysis to predict the mechanisms of action for inhibitor–kinase interactions that can facilitate the development of next-generation PKC modulators.
Protein kinases are a large and diverse family of more than 500 proteins encoded by ~2% of the human genome. Kinases regulate signaling networks by catalyzing the phosphorylation of specific amino acids with adenosine triphosphate (ATP) as the phosphate source, resulting in a conformational change from an inactive to an active form of the substrate or from an active to inactive conformation. Approximately one-third and up to two-thirds of the proteins in a cell may be phosphorylated at one time or another, affecting a very large set of cellular pathways by turning activities “on” or “off” [1,2]. Phosphorylation plays major roles in numerous cellular functions, including transcription, translation, metabolism, proliferation, division, cell-cycle progression, biosynthesis, movement, and survival [3]. These processes are critical to cellular homeostasis, and dysregulated kinase activity has been linked to a variety of pathological conditions, such as neurodegeneration [4], inflammation [5], autoimmunity [5], cancer [6,7,8], and cardiovascular diseases (CVDs) [9]. Imatinib (i.e., STI571, or Gleevec), which received Food and Drug Administration (FDA) approval in 2001, is considered the first protein kinase inhibitor that was clinically approved [10]. Although fasudil (an inhibitor of Rho-dependent kinases) was approved in 1995, and rapamycin (i.e., sirolimus, an inhibitor of the protein kinase TORC1) was approved in 1999, both were approved without the knowledge of the identity of their target proteins. Imatinib is a potent small-molecule kinase inhibitor (SMKI) treatment for chronic myelogenous leukemia (CML), that functions as a competitive inhibitor of the ATP-binding site. Imatinib has revolutionized drug therapy for CML, and the drug was featured on the front cover of Time Magazine (28 May 2001, Vol. 157 No. 21), termed as a “magic bullet”.
Since phosphorylation plays major roles in numerous cellular functions, and it is critical to cellular homeostasis, it is not surprising that protein kinases are the second most therapeutically targeted group of proteins, after the G-protein-coupled receptors (GPCRs), and the pharmaceutical industry has dedicated approximately one-third of new drug development programs over the last decade to the development of protein kinase modulators [11,12]. Currently, there are 98 approved kinase inhibitors worldwide, 71 of which are SMKIs that have been approved by the FDA, targeting 21 kinase families constituting approximately 20% of the kinome. Interestingly, the number of SMKIs approved by the FDA has more than doubled since 2016, with 37 new approvals, making SMKIs approximately 15% of all novel drug approvals in the last 5 years (2016–2021). In addition, 16 more SMKIs have been granted approval by other regulatory agencies [13]. Currently, the majority of the FDA-approved kinase inhibitors are SMKIs targeting the kinase ATP-binding site (63 SMKIs) [14]. However, in many cases, these SMKIs demonstrate low specificity towards the target kinase, resulting in problems with toxicity and a variety of side effects, and is a major cause of clinical trial failure. Therefore, the pharmaceutical industry continues to invest in emerging trends focused on identifying alternative approaches to specifically target protein kinases more selectively.
While the majority of studies on protein kinases focused on their catalytic activity, others demonstrated that their noncatalytic properties are also indispensable, and, in some cases, even sufficient for their effector function. Over 25 years ago, the yeast Pbs2p protein was found to serve both as a protein kinase and a scaffold protein [15]. Since then, accumulated evidence suggests that kinases possess functions beyond catalysis (noncatalytic functions), such as the scaffolding of protein complexes, allosteric regulation of other proteins through protein–protein interactions (PPIs), subcellular targeting, and deoxyribonucleic acid (DNA) binding. This diverse spectrum of activities can be used to coordinate substrate phosphorylation in a highly specific manner and support other functions that do not rely on kinase activity [16]. These noncatalytic activities involve unique interaction sites that are not as conserved as the phosphorylation site. Therefore, blocking these functions by targeting less conserved binding sites that mediate noncatalytic functions may support greater selectivity, thus reducing off-target effects [17]. These allosteric modulators also achieve high selectivity by targeting inactive kinase conformations, in which the structure does not need to be catalytically competent. Thus, each protein kinase may be targeted in a unique conformation, providing far greater opportunities for selectivity [18,19].
The allosteric modulation approach has become an important one in drug discovery for the development of compounds that bind to sites distinct from the conserved ATP-binding site. Allosteric modulators have been used to target all major mammalian receptor superfamilies, including GPCRs, ligand-gated ion channels, and intracellular nuclear hormone receptors, providing new opportunities for basic research, as well as for therapeutic application [20,21,22]. These sites that are less conserved across the kinome and many times only available upon conformational changes provide several advantages, particularly, higher selectivity and extended drug target residence times [23]. For example, Chaikuad et al. developed the small molecule SCH772984, which is a highly specific inhibitor of extracellular signal-regulated kinase (Erk) 1 (Erk1) and Erk2. The compound targets an allosteric site, which was a previously unidentified binding pocket. The same inhibitor also binds in a completely different conformation with a lower affinity to off-target kinases [24]. An allosteric approach to target protein kinase was used by Zorba et al. for the development of monobodies (small proteins) to act either as kinase inhibitors or activators via the differential recognition of structural motifs in the allosteric pocket of the oncoprotein Aurora A (AurA) kinase. These investigators solved the crystal structure of AurA bound to activating and inhibiting monobodies, shedding light on the mechanism underlying allosteric modulation [25].
Many approved SMKIs demonstrate a low selectivity profile, directing the community to investigate less conserved non-ATP-binding sites. To address this issue, allosteric inhibitors targeting sites other than the orthosteric ATP-binding pocket have been developed. Allosteric kinase modulators are inhibitors that bind to an allosteric site outside the conserved ATP-binding pocket with no direct interaction with the hinge region of the ATP-binding domain, providing a significant opportunity for the generation of new classes of highly selective kinase regulators [26]. The field of allosteric kinase inhibition has evolved rapidly in the past few years with the FDA approval of the first allosteric kinase inhibitor trametinib (2018) (i.e., mekinist or meqsel). Trametinib (GSK1120212) is a unique reversible selective orally bioavailable (a mitogen-activated protein kinase (MAPK)/ERK kinase) MEK allosteric inhibitor with high affinity and nanomolar activity, which specifically binds to MEK1 and MEK2. Trametinib acts as a non-competitive ATP inhibitor that stably binds to unphosphorylated MEK and, thereby, suppresses the downstream signaling pathways involved in cell proliferation, survival, and differentiation. Trametinib demonstrated several advantages over other inhibitors, such as an improved half-life, limited toxicity, and limited interaction with other drugs. Trametinib was approved in 2013 by the FDA for the treatment of patients with V600E mutated metastatic melanoma [27].
Protein–protein interactions (PPIs) represent a significant portion of functionally relevant biological interactions and are central to most biological processes [28]. Current estimates suggest that the human repertoire of PPIs (the interactome) ranges from 130,000 to 600,000 interactions [29]. PPIs are often dysregulated in human diseases and, therefore, represent a rich source of potential therapeutic targets. Targeting PPI sites offers the potential to differentiate between many proteins and even homologous enzymes, since the sequence and/or structure of these sites are usually unique [30,31]. Targeting PPIs with small molecules is challenging, as the binding surfaces between proteins are usually large and flat and involve polar and hydrophobic interactions without a defined binding pocket. Furthermore, small molecules usually demonstrate low specificity, resulting in toxicity. Targeting PPIs with antibodies is also not straightforward, as their production can be difficult and expensive, they have low oral bioavailability, and they are usually not cell-permeable [32,33]. On the other hand, peptides and peptidomimetics (modified peptides, a term henceforth used interchangeably with “peptides” due to the overlap in defining these species) are ideal candidates to target PPIs for their unique properties, as discussed below [34,35,36,37,38].
Peptides are especially useful candidates for the inhibition of PPIs because they can mimic a protein surface to effectively compete for binding. Peptides demonstrate many advantages for targeting protein complexes compared to small molecules, such as conformational flexibility [39] and increased selectivity [40,41], thereby improving drug properties and limiting toxicity. In addition, peptides are easier and less expensive to manufacture compared to antibodies [42]. In many cases, the number of amino acids that form the PPI site and govern the binding is small (only few amino acids) [43], and it is estimated that between 15–40% of all PPIs in the cell are regulated by short linear peptides [44]. Importantly, short linear peptides derived from PPI sites can mimic the interaction site on one protein, serving as competitive inhibitors or antagonists of the respective interaction [45,46].
Peptide therapeutics have played a notable role in medical practice since the isolation and commercialization of insulin [47], which was the first peptide to be administered therapeutically. Currently, peptides are used for a wide range of indications, including metabolic disease, infectious disease, neurological disease, autoimmune diseases, oncology, CVDs, and a variety of other disorders [48,49]. The number of peptides entering clinical studies continues to grow, from 1.2 per year (1970s) to over 16.8 per year (2000s) [50,51]. Peptides demonstrate superior success rates in transitioning from phase 1 to phase 2 trials (83%) compared to small molecules and biological drugs (63% and 77%, respectively) and in transitioning from phase 3 to regulatory review (68% compared to 61% for small molecules and 63% for biological drugs) [52]. Aside from crossing the cell membrane independently (e.g., cyclosporine), peptides and peptidomimetics can be conjugated to cell-penetrating vehicles to modulate intracellular targets [53]. The coupling of cargo to cell-permeable peptides has been used extensively to deliver molecules, peptides, and proteins in cells [54], animal models [55], and humans [56] (for a review, see [57]). It is not surprising that the number of available therapeutic peptides is increasing, and, as of 2020, there were >100 approved peptides and peptidomimetics with therapeutic or diagnostic applications on the market. About 155 peptides are in clinical trials, and over 500 are in preclinical development. In addition, four peptides reached global sales of over $1 billion as early as 2010, including glatiramer acetate ($4.0 billion), leuprolide acetate ($3.0 billion), octreotide acetate ($1.3 billion), and goserelin acetate ($1.1 billion) [58,59,60,61].
There are several approaches to identify peptides that target PPIs, including rational design methods, as well as screening large peptide libraries in which peptides can be designed using systematic or random methodologies. The major approaches that use large screen methodologies include the orderly search of large domains involved in PPIs, random search of large domains involved in PPIs, and search of key amino acid residues involved in PPIs. While all the above approaches were used successfully to identify peptides that regulate PPIs, they are labor-intensive and costly, as they require the use of large libraries, limiting their practical use. Herein, we will discuss several rational approaches to develop peptides that target kinase PPIs by allosteric modulation.
