Today’s post is written by Michael Curtin, Senior Product Manager, Reporters and Signaling.
Inflammation is a defense mechanism that the body employs in which the immune system recognizes and removes harmful and foreign stimuli and begins the healing process. Inflammation can be either acute or chronic. Chronic inflammation is also referred to as slow, long-term inflammation and can last for prolonged periods (several months to years); chronic inflammation is caused by immune dysregulation. This typically takes the form of the body’s inability to resolve inflammation resulting from overproduction of inflammatory cytokines and chemokines, as well as danger-associated molecular patterns (DAMPs) released from dying cells (2). Tumor Necrosis Factor (TNF) is the primary cytokine involved in many common inflammatory diseases and is where many therapies targeting inflammation are focused.
Recent research that RIP kinases (RIPK1 and RIPK3) are important regulators of innate immunity via their key roles in cell death signaling during cellular stress and following exposure to inflammatory and infectious stimuli. RIPK1 has an important scaffolding role in pro-inflammatory signaling where it interacts with TRADD, TRAF1 TRAF2, and TRAF3 and TRADD can act as an adaptor protein to recruit RIPK1 to the TNFR1 complex in a TNF-dependent process. RIPK1 plays a kinase activity-dependent role in both apoptotic and necroptotic cell death. A review article by Speir et al. (1) discusses the role of RIP kinases in chronic inflammation and the potential of RIPK1 inhibitors as a new therapeutic approach for the treatment of chronic inflammation. RIPK1 or Receptor Interacting Protein Kinase 1 is a serine/threonine kinase that was originally identified as interacting with the cytoplasmic domain of FAS. Promega offers several reagents that make studying RIPK1 easier- these include our RIPK1 Kinase Enzyme Systems which includes RIPK1 (Human, recombinant; amino acids 1-327), myelin basic protein (MBP) substrate, reaction buffer, MnCl2, and DTT and is optimized for use with our ADP-Glo Kinase Assay.
Antibody-based immune checkpoint inhibitors remain a major focus of immuno-oncology drug research and development efforts because of their recent success in providing long-term anti-tumor responses. However, the range of response of different tumor types to these drugs is hugely varied. Small molecule kinase inhibitors that block signaling pathways involved in regulation of tumor immunity at multiple points in the “cancer immunity cycle” may provide alternate, effective therapeutics. One kinase that may be a target for such small molecule inhibitors is Hematopoietic Progenitor Kinase 1 or HPK1; the potential of this kinase as a therapeutic target was reviewed by Sawasdikosol and Burakoff (1). HPK1, also known as MAP4K1, is a member of the MAP kinase protein kinase family that negatively regulates signal transduction in T-cells, B-cells and dendritic cells of the immune system.
Cyclin-dependent kinases (CDKs) are promising therapeutic targets in cancer and are currently among the most intensely studied enzymes in drug discovery. The FDA has recently approved three drugs for breast cancer that target members of this kinase subfamily, fueling interest in the entire family. Although broad efforts in drug discovery have produced many CDK inhibitors (CDKIs), few have been characterized in living cells. So just how potent are these compounds in a cellular environment? Are these compounds selective for their intended CDK target, or do they bind many similar kinases in cells? To address these questions, teams at the Structural Genomics Consortium and Promega used the NanoBRET™ Target Engagement technology to uncover surprising patterns of selectivity for touted CDKIs and abandoned clinical leads (1). The results offer exciting opportunities for repurposing some inhibitors as selective chemical probes for lesser-studied CDK family members.
CDKs and CDKIs
Cyclin-dependent kinases (CDKs) regulate a number of key global cellular processes, including cell cycle progression and gene transcription. As the name implies, CDK activity is tightly regulated by interactions with cyclin proteins. In humans, the CDK subfamily consists of 21 members and several are validated drivers of tumorigenesis. For example, CDKs 1, 2, 4 and 6 play a role in cell cycle progression and are validated therapeutic targets in oncology. However, the majority of the remaining CDK family is less studied. For example, some members of the CDK subfamily, such as CDKs 14–18, lack functional annotation and have unclear roles in cell physiology. Others, such as the closely related CDK8/19, are members of multiprotein complexes involved broadly in gene transcription. How these kinases function as members of such large complexes in a cellular context remains unclear, but their activity has been associated with several pathologies, including colorectal cancer. Despite their enormous therapeutic potential, our knowledge of the CDK family members remains incomplete.
The understudied kinome represents a major challenge as well as an exciting opportunity in drug discovery. A team of researchers lead by Nathanael Gray at the Dana Farber Cancer Institute was able to partially elucidate the function of an understudied kinase, Doublecortin-like kinase 1 (DCLK1), in pancreatic ductal adenocarcinoma cells (PDAC). The characterization of DCLK1 in PDAC was realized by developing a highly specific chemical probe (1). Promega NanoBRET™ Target Engagement (TE) technology enabled intracellular characterization of this chemical probe.
The Dark Kinome
Comprised of over 500 proteins, the human kinome is among the broadest class of enzymes in humans and is rife with targets for small molecule therapeutics. Indeed, to date, over 50 small molecule kinase inhibitors have achieved FDA approval for use in treating cancer and inflammatory diseases, with nearly 200 kinase inhibitors in various stages of clinical evaluation (2). Moreover, broad genomic screening efforts have implicated the involvement of a large fraction of kinases in human pathologies (3). Despite such advancements, our knowledge of the kinome is limited to only a fraction of its family members (3,4). For example, currently less than 20% of human kinases are being targeted with drugs in clinical trials. Moreover, only a subset of kinases historically has garnered substantial citations in academic research journals (4). As a result, a large proportion of the human kinome lacks functional annotation; as such, these understudied or “dark” kinases remain elusive to therapeutic intervention (4).
The review “Kinase Inhibitors: the road ahead” was recently published in Nature Reviews Drug Discovery. In it, authors Fleur Ferguson and Nathanael Gray provide an up-to-date look at the “biological processes and disease areas that kinase-targeting small molecules are being developed against”. They note the related challenges and the strategies and technologies being used to efficiently generate highly-optimized kinase inhibitors.
This review describes the state of the art for kinase inhibitor therapeutics. To understand why kinase inhibitors are so important in the development of cancer (and other) therapeutics research, let’s start with the role of kinases in cellular physiology.
Drug research and development is a complex and expensive process that begins with initial screening steps of candidate chemical compounds, and compounds that appear to have the desired potency against a specific cellular target or pathway are further evaluated. Candidate compounds that fail late in development or during clinical trials because of off-target effects are costly, and can be dangerous. Therefore drug developers not only need to ensure that a candidate compound is effective as a therapy, but also they need to predict any potential undesirable side effects due to off-target activities as early as possible in the drug discovery and development process. Continue reading “Making Drug Discovery More Efficient: Predicting Drug Side Effects in Early Screening Efforts”
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