kinase screening

HPK1 Identified as Emerging Immuno-oncology Drug Target

Posted on by Michele Arduengo

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.

Artist rendering of what target engagement might look like for kinases like HPK1.
NanoBRET™ Target Engagement Assay (artist rendering)
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The Surprising Landscape of CDK Inhibitor Selectivity in Live Cells

Posted on by Promega

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

nanobret technology for kinase target engagement

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.

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Illuminating the Function of a Dark Kinase (DCLK1) with a Selective Chemical Probe

Posted on by Promega

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

NanoBRET target engagement

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).

Continue reading “Illuminating the Function of a Dark Kinase (DCLK1) with a Selective Chemical Probe”

Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action

Posted on by Michele Arduengo

Fig 4. Four point MMOA screen for tideglusib and GW8510. Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.
Four point MMOA screen for tideglusib and GW8510.
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature.  Black = 30 min preincubation Grey = No preincubation.
The first small-molecule kinase inhibitor approved as a cancer therapeutic, imatinib mesylate (Gleevec® treatment), has been amazingly successful. However, a thorough understanding of its molecular mechanism of action (MMOA) was not truly obtained until more than ten years after the molecule had been identified.

Understanding the MMOA for a small-molecule inhibitor can play a major role in optimizing a drug’s development. The way a drug actually works–the kinetics of binding to the target molecule and how it competes with endogenous substrates of that target–ultimately determines whether or not a a candidate therapeutic can be useful in the clinic. Drugs that fail late in development are extremely costly.

Drug research and discovery for neglected tropical diseases suffer from a lack of a large commercial market to absorb the costs of late-stage drug development failures. It becomes very important to know as much as possible, simply and quickly, about MMOA for candidate molecules for these diseases that are devastating to large populations.

One such neglected topical disease is Human African trypanosomiasis (HAT, also known as sleeping sickness). Continue reading “Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action”

Shining a Bright Light on Deep Questions in Biology with Bioluminescence

Posted on by Michele Arduengo
artists view inside a cell

Search the PubMed database for “dual-luciferase” and citations abound. The Dual-Luciferase® Reporter Assay is a powerful tool that allows researchers to ask a multitude of questions about gene control and expression in a system that itself could be normalized and internally controlled. For more than 15 years, firefly and Renilla luciferases  have formed the basis of a range of powerful assays and research tools for scientists who are asking questions about the deep and complex genetic and cellular story associated with cancer. Here we talk a bit of about bioluminescent chemistries, some of the newest bioluminescent tools available, and how some of these tools can be used to probe the deeper questions of cell biology, including cancer biology.

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