GPCRs and PROTACs: New Approaches for Designing More Effective Drug Candidates

NanoBRET target engagement assay

G protein-coupled receptors (GPCRs) comprise a large group of cell surface receptors, characterized by the unique structural property of crossing the cell membrane seven times. They respond to a diverse group of signaling molecules, such as peptides, neurotransmitters, cytokines, hormones and other small molecules (1). Upon activation, GPCRs interact with GTP-binding (G) proteins and arrestins to regulate a wide variety of signaling pathways. This broad range of functions makes GPCRs attractive targets for drug discovery. The importance of GPCR research was highlighted in 2012, with the Nobel Prize in chemistry being awarded to Robert Lefkowitz and Brian Kobilka “for studies of G-protein–coupled receptors”.

Based on structure and function, GPCRs are categorized into six classes, A–F. The class A GPCRs, or rhodopsin-like receptors, have been studied extensively due to their association with many types of diseases (2). Within the class A GPCRs is a group that share a highly conserved structural motif (3) and respond to chemokines—small “chemotactic cytokines” that stimulate cell migration, especially that of white blood cells (4). A subfamily of class A GPCRs respond to chemokines that have two cysteine residues near the N-terminus, known as CC chemokines. GPCRs activated by CC chemokines are called CC chemokine receptors or CCRs, and these interactions have been implicated in both pro- and anti-cancer pathways (5).

Continue reading “GPCRs and PROTACs: New Approaches for Designing More Effective Drug Candidates”

The Surprising Landscape of CDK Inhibitor Selectivity in Live Cells

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.

Continue reading “The Surprising Landscape of CDK Inhibitor Selectivity in Live Cells”

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

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”

NanoLuc® Luciferase Powers More than Reporter Assays

Bright NanoLuc® Luciferase

What can you do with a small, super bright luciferase? Amazing things. We’ve highlighted many of the papers and new applications that NanoLuc® luciferase has enabled on this blog. While NanoLuc® luciferase was first introduced as a reporter enzyme to assess promoter activity, its capabilities have expanded far beyond a genetic reporter, creating bioluminescent tools used to study endogeneous protein dynamics, target engagement, protein degradation, immunodetection and more. So where did the NanoLuc luciferase come from and how does one enzyme power so many research capabilities? Read further for a primer on the various technologies and applications developed from this enzyme over the last 10 years.

Continue reading “NanoLuc® Luciferase Powers More than Reporter Assays”

Voted Drug Discovery and Development Product for 2018: NanoBRET TE Kinase Assays

Choice Drug Discovery and Development Product 2019 award
Michael Curtin, Promega, accepting the Reviewers’ Choice for Drug Discovery and Development Product of the Year award from SelectScience.

As announced at SLAS in Washington, D.C. recently, we are excited to have NanoBRET Target Engagement (TE) Intracellular Kinase Assays awarded the SelectScience Reviewers’ Choice for Drug Discovery and Development Product of the Year 2018!

The NanoBRET™ Target Engagement (TE) Kinase Assay, first available in the fall of 2017, has been getting great reviews on the SelectScience site for more than a year now. Continue reading “Voted Drug Discovery and Development Product for 2018: NanoBRET TE Kinase Assays”

Quantitating Kinase-Inhibitor Interactions in Live Cells

Kinase target engagement is a new way to study kinase inhibitors for target selectivity, potency and residency. The NanoBRET™ TE Intracellular Kinase Assays enable you to quantitate kinase-inhibitor binding in live cells, making these assays an exciting new tool for kinase drug discovery research.

For today’s blog about NanoBRET™ TE Intracellular Kinase Assay, we feature spokesperson Dr. Matt Robers. Matt is part of Promega’s R & D department and is one of the developers of the NanoBRET™ TE Intracellular Kinase Assay. Continue reading “Quantitating Kinase-Inhibitor Interactions in Live Cells”

Kinase Drug R & D: Helping Your Inhibitor Make the Cut

Finding the best inhibitor for your kinase doesn’t have to be a long trip.

A recent paper in Journal of Medicinal Chemistry, “Discovery of GDC-0853: A Potent, Selective and Noncovalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development” (1) details some elegant work in chemical modification and extensive testing during exploration of inhibitors for BTK. As a warmup to the article, here is a brief BTK backstory.

BTK (Bruton Tyrosine Kinase): Importance in Health and Disease 

Bruton’s tyrosine kinase (BTK) was initially identified as a mediator of B-cell receptor signaling in the development and functioning of adaptive immunity. More recent and growing evidence supports an additional role for BTK in mononuclear cells of the innate immune system, especially dendritic cells and macrophages. For example, BTK functions in receptor-mediated recognition of infectious agents, cellular maturation and recruitment processes, and Fc receptor signaling. BTK has recently been identified as a direct regulator of a key innate inflammatory machinery, the NLRP3 inflammasome (2). Continue reading “Kinase Drug R & D: Helping Your Inhibitor Make the Cut”

Kinase Inhibitors as Therapeutics: A Review

This blog was originally published in April of 2018. This update includes the paper, “Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement” from Cell Chemical Biology, demonstrating the power of NanoBRET™ target engagement kinase assays in the study of kinase inhibitors.

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.

The road ahead for kinase inhibitor studies.

Why Kinases? Continue reading “Kinase Inhibitors as Therapeutics: A Review”

NanoBRET™ Target Engagement Intracellular Kinase Assay Nominated for Scientists’ Choice Award®

Joins Nominees for Best New Drug Discovery & Development Product 2017

SelectScience® nominates NanoBRET™ Target Engagement Kinases Assay as a Best New Drug Discovery & Development product for 2017.

We were honored recently to have NanoBRET™ Target Engagement Intracellular Kinase Assays nominated by SelectScience® as one of the Best New Drug Discovery & Development Products of 2017. This is a Scientists’ Choice Award®, an opportunity for scientists like you worldwide to vote for your favorite new drug discovery/development product.

We are super excited about both the nomination and the NanoBRET™ Target Engagement Intracellular Kinase Assay. Here is a little information about the assay.

Continue reading “NanoBRET™ Target Engagement Intracellular Kinase Assay Nominated for Scientists’ Choice Award®”

Your New Best Research Partner: The Structural Genomics Consortium

Research surrounding drug discovery has historically been highly competitive and expensive. Unfortunately, many late-stage drug failures have occurred over recent years, often due to lack of efficacy. These failures have left the industry searching for new means by which to improve early drug discovery efforts aimed at understanding the drug target and its role in disease. One idea that is gaining traction is partnerships to openly share information at the early, precompetitive stages of drug discovery.

I used to think of open access only in terms of publishing data and information—online sites where you could freely access data without a subscription or membership, and without payment.

Structural Genomics Consortium logo.

Meet the Structural Genomics Consortium (SGC), the international partnership that’s taking open access to a new level in order to advance scientific research for scientists working in a variety of disciplines—structural genomics and beyond. The SGC might just become your new, best laboratory research partner. Continue reading “Your New Best Research Partner: The Structural Genomics Consortium”