Elsevier

Pharmacological Research

Volume 100, October 2015, Pages 1-23
Pharmacological Research

Invited Review
A historical overview of protein kinases and their targeted small molecule inhibitors

https://doi.org/10.1016/j.phrs.2015.07.010Get rights and content

Abstract

Protein kinases play a predominant regulatory role in nearly every aspect of cell biology and they can modify the function of a protein in almost every conceivable way. Protein phosphorylation can increase or decrease enzyme activity and it can alter other biological activities such as transcription and translation. Moreover, some phosphorylation sites on a given protein are stimulatory while others are inhibitory. The human protein kinase gene family consists of 518 members along with 106 pseudogenes. Furthermore, about 50 of the 518 gene products lack important catalytic residues and are called protein pseudokinases. The non-catalytic allosteric interaction of protein kinases and pseudokinases with other proteins has added an important regulatory feature to the biochemistry and cell biology of the protein kinase superfamily. With rare exceptions, a divalent cation such as Mg2+ is required for the reaction. All protein kinases exist in a basal state and are activated only as necessary by divergent regulatory stimuli. The mechanisms for switching between dormant and active protein kinases can be intricate. Phosphorylase kinase was the first protein kinase to be characterized biochemically and the mechanism of its regulation led to the discovery of cAMP-dependent protein kinase (protein kinase A, or PKA), which catalyzes the phosphorylation and activation of phosphorylase kinase. This was the first protein kinase cascade or signaling module to be elucidated. The epidermal growth factor receptor-Ras-Raf-MEK-ERK signaling module contains protein-tyrosine, protein-serine/threonine, and dual specificity protein kinases. PKA has served as a prototype of this enzyme family and more is known about this enzyme than any other protein kinase. The inactive PKA holoenzyme consists of two regulatory and two catalytic subunits. After binding four molecules of cAMP, the holoenzyme dissociates into a regulatory subunit dimer (each monomer binds two cAMP) and two free and active catalytic subunits. PKA and all other protein kinase domains have a small amino-terminal lobe and large carboxyterminal lobe as determined by X-ray crystallography. The N-lobe and C-lobe form a cleft that serves as a docking site for MgATP. Nearly all active protein kinases contain a K/E/D/D signature sequence that plays important structural and catalytic roles. Protein kinases contain hydrophobic catalytic and regulatory spines and collateral shell residues that are required to assemble the active enzyme. There are two general kinds of conformational changes associated with most protein kinases. The first conformational change involves the formation of an intact regulatory spine to form an active enzyme. The second conformational change occurs in active kinases as they toggle between open and closed conformations during their catalytic cycles. Because mutations and dysregulation of protein kinases play causal roles in human disease, this family of enzymes has become one of the most important drug targets over the past two decades. Imatinib was approved by the United States FDA for the treatment of chronic myelogenous leukemia in 2001; this small molecule inhibits the BCR-Abl protein kinase oncoprotein that results from the formation of the Philadelphia chromosome. More than two dozen other orally effective mechanism-based small molecule protein kinase inhibitors have been subsequently approved by the FDA. These drugs bind to the ATP-binding site of their target enzymes and extend into nearby hydrophobic pockets. Most of these protein kinase inhibitors prolong survival in cancer patients only weeks or months longer than standard cytotoxic therapies. In contrast, the clinical effectiveness of imatinib against chronic myelogenous leukemia is vastly superior to that of any other targeted protein kinase inhibitor with overall survival lasting a decade or more. However, the near universal and expected development of drug resistance in the treatment of neoplastic disorders requires new approaches to solve this therapeutic challenge. Cancer is the predominant indication for these drugs, but disease targets are increasing. For example, we can expect the approval of new drugs inhibiting other protein kinases in the treatment of illnesses such as hypertension, Parkinson's disease, and autoimmune diseases.

Section snippets

The protein kinase enzyme family

Protein kinases play pivotal roles in nearly every aspect of cellular function [1]. They control metabolism, transcription, cell division and movement, programmed cell death, and they participate in the immune response and nervous system function. Protein phosphorylation involves the balanced action of protein kinases and phosphoprotein phosphatases making phosphorylation–dephosphorylation an overall reversible process [1], [2]. Owing to the overall importance of protein phosphorylation,

Phosphorylated proteins

Casein, a milk protein, and phosvitin, an egg yolk protein, are two of the earliest known phosphoproteins [10]. Casein contains about 3% and phosvitin contains about 10% phosphorus by weight. The latter contains one phosphate group for every two amino acid residues thereby making it the most highly phosphorylated protein in nature. Lipmann and Levine identified phosphoserine in phosvitin in 1932 [10]. At the time threonine was unknown. W.C. Rose and two of his graduate students described it as

Protein kinases and their activation by second messengers

Working in the Ben May Laboratory for Cancer Research at the University of Chicago, Williams-Ashman and Kennedy [16] reported that protein phosphorylation was especially active in malignant cells such as Ehrlich ascites tumor cells. Subsequently, Kennedy and Smith isolated radioactive phosphoserine of very high specific activity from the protein fraction of these tumor cells after incubation with [32P]-phosphate [17]. They demonstrated that the phosphate moiety of phosphoserine in the protein

Primary structures of protein kinases

Czernilofsky et al. [66], [67] reported the amino acid sequence of the Schmidt-Rupin strain of v-Src in 1980 based upon its nucleotide sequence and Shoji et al. [68] reported the sequence of the catalytic subunit of bovine PKA in 1981 using Edman degradation of cyanogen bromide and trypsin peptides. Owing to the incomplete nature of the protein sequence data bases at the time, the identification of v-Src as a protein kinase was not made until 1982 [69]. The signatures that enabled this

The regulatory spine

Kornev et al. [81], [82] compared the spatial arrangements of amino acid residues in about two dozen active and inactive protein kinases using a local spatial pattern (LSP) alignment algorithm. They used this information to establish the existence of a regulatory and a catalytic spine within the protein kinase domain. In contrast to protein kinase amino acid signatures such as Y/HRD or DFG, the residues that constitute the spines were not identified by sequence analyses per se. Rather, the

PKA signaling

As noted previously, the PKA holoenzyme consists of two general types (I and II) originally based upon their order of elution from DEAE-cellulose anion exchange resin; the type I enzyme elutes first at a lower salt concentration than the type II enzyme. As noted previously, PKA holoenzymes exist as an inactive tetrameric complex composed of two catalytic and two regulatory subunits (R2C2) [60]. Mammals possess four non-redundant R-subunits (RIα, RIβ, RIIα, and RIIβ), which differ in their

Secondary and tertiary structures of Abl

The small lobe of all protein kinases including Abl is dominated by a five-stranded antiparallel β-sheet (β1–β5) and an important regulatory αC-helix [74], [98]. The first X-ray structure of a protein kinase (PKA) [72], [73] contained an αA and an αB-helix proximal to αC (PDB ID:2CPK), but these first two helices are not conserved in the protein kinase family. The large lobe of the Abl protein kinase domain is mainly α-helical with six conserved segments (αD–αI) that occur in all protein

Pseudokinase properties

In their comprehensive description of the protein kinase complement of the human genome (kinome), Manning et al. [1] reported that 50 of the 518 protein kinases lack amino acid residues important for catalysis. Thus, the absence of the αC-Glu or β3-Lys or alterations in the DFG or HRD signatures was ascribed to catalytically inactive kinases, or pseudokinases. These pseudokinases are scattered throughout the various protein kinase subfamilies, suggesting that they have evolved from diverse

Overview of inhibitors

Because mutations and dysregulation of protein kinases play causal roles in human disease, this family of enzymes has become one of the most important drug targets over the past two decades [108]. Trastuzumab was the first FDA-approved protein kinase inhibitor (1999); this biologic is a monoclonal antibody that inhibits ErbB2 and is used for the treatment of ErbB2-positive breast, gastric, and gastroesophageal cancers [45], [77]. Several other large molecule protein kinase inhibitors have been

Ph+ chronic myelogenous leukemia

Rowley discovered that the Philadelphia chromosome is formed from a reciprocal translocation t(9;22)(q34;q11.2) that results in a lengthened chromosome 9 and a shortened chromosome 22 (the Philadelphia chromosome) [132]. The Philadelphia chromosome occurs in about 95% of chronic myelogenous leukemia patients. The BCR-Abl oncogene results from this translocation where Abl is the human ortholog of the murine Abelson leukemia virus, which was originally on chromosome 9, and BCR refers to the

The role of serendipity in drug development

Imatinib was developed as a PDGFR inhibitor, but its initially approved therapeutic targets included BCR-Abl and Kit [109], [164]. Imatinib was approved in 2001 for the treatment of BCR-Abl-positive CML and in 2002 for the treatment of GIST with KIT mutations. The drug has subsequently been FDA-approved for the treatment of a variety of other diseases including adults with (i) relapsed or refractory Ph+ ALL, (ii) myelodysplastic/myeloproliferative diseases associated with PDGFR gene

Epilog

Human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) result in a disease with variable signs and symptoms [180]. HIV is a retrovirus that primarily infects components of the human immune system such as CD4+ T cells, macrophages, and dendritic cells. It directly and indirectly destroys CD4+ T cells. HIV1 and HIV2 contain genes that encode the proteins required for virus replication: gag encodes the proteins that form the core of the virion, pol encodes

Conflict of interest

The author is unaware of any affiliations, memberships, or financial holdings that might be perceived as affecting the objectivity of this review.

Acknowledgement

The author thanks Laura M. Roskoski for providing editorial and bibliographic assistance.

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