Small-Molecule Drug Discovery

Residence Time: The Impact of Binding Kinetics on Compound-Target Interactions

Posted on by Promega

This blog was written by guest contributor Tian Yang, Associate Product Manager, Promega, in collaboration with Kristin Huwiler, Manager, Small Molecule Drug Discovery, Promega.

During the development of chemical probes or small-molecule drugs, compound affinity (Kd) or potency (IC50) is used to characterize compound-target interactions, to guide structure-activity relationship analysis and lead optimization and to assess compound selectivity.

However, neither parameter provides information on how quickly a compound engages with and dissociates from the target. The dissociation constant Kd reflects the relative concentrations of unbound and bound state of the compound at thermodynamic equilibrium, and while IC50 is an empirical metric that measures the concentration at which a biochemical or cellular process is reduced to half of the maximum value, IC50 values are typically determined when the process is assumed to be at equilibrium or steady-state. For a closed system, like cells in a culture dish, these thermodynamic parameters are quite informative. In an open system like the human body, where compound-target interactions often do not reach equilibrium, the kinetic parameters, in addition to the thermodynamic parameters, are needed to better understand and characterize compound target engagement over time (1,2).

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Measure Engagement to Target Proteins within Complexes: Why Context Matters

Posted on by Promega

This blog was written by guest contributor Tian Yang, Associate Product Manager, Promega, in collaboration with Kristin Huwiler, Manager, Small Molecule Drug Discovery, Promega.

For target-based drug discovery programs, biochemical assays using purified target proteins are often run for initial hit discovery, as these assays are target-specific, quantitative and amenable for high-throughput screens, allowing for precise characterization of target-compound interactions. However, proteins do not act in isolation inside the cells. Instead, proteins form complexes with other cellular components to drive cellular processes, signaling cascades, and metabolic pathways. Just as the interactions between a target protein and its binding partners can influence the target function, compound engagement with target proteins can vary depending on the protein complex formed.

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Exploring the Relationship Between IC50 and Kd in Pharmacology

Posted on by Promega

This guest blog post is written by Tian Yang, Associate Product Manager at Promega.

In the realm of chemical probe development and drug discovery, understanding the interactions between drugs/compounds and their targets is crucial. Two frequently used metrics to characterize these interactions are IC50 and Kd, which guide researchers in evaluating the potential of compounds in effecting changes in target function. IC50 offers insights into a compound’s potency by quantifying its ability to inhibit a specific biological activity. Kd provides a measure of the affinity between a ligand and its receptor, reflecting how tightly a compound binds to its target (1). Together, these parameters are instrumental in the early stages of drug development, helping to identify promising candidates by assessing a compounds’s binding characteristics and its observed efficacy.

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Drug Target Confirmed? Tivantinib’s Lesson on the Importance of Cellular Target Engagement

Posted on by Promega

This guest blog post is written by Tian Yang, Associate Product Manager at Promega.

There are often challenges with translating results from a test tube into a living system, demanding more physiologically relevant assays. In drug discovery, demonstrating a compound’s ability to modulate its target protein in live cells is a critical step in the hit-to-lead workflow. A variety of cell-based assays can be used to assess a compound’s activity in live cells. Take kinase inhibitors as an example, these assays can range from substrate phosphorylation assays that more directly report on the activity of target kinases, to genetic reporter assays or cell viability assays that assess the downstream effects of target modulation.

In the case of Tivantinib, several pieces of data from its development were used to establish its role as an inhibitor of MET kinase. MET Kinase is a prominent target for anti-cancer therapeutics due to frequent MET dysregulation in a wide range of tumors. For example, over-activation of MET drives cancer proliferation and metastasis. In the initial report on Tivantinib, in addition to biochemical activity assays performed with purified MET, the activity of Tivantinib in cells was verified by several methods, including: 1) inhibition of phosphorylation of MET and downstream signaling pathways, 2) cytotoxicity in cancer cell lines expressing MET, and 3) antitumor activity in xenograft mouse models (1). Additionally, a co-crystal structure of the MET-Tivantinib complex was solved, seemingly confirming that Tivantinib is a bona fide MET inhibitor capable of engaging MET in live cells (2). Based on these observations and other pre-clinical data, Tivantinib appeared to be a promising drug candidate and was taken through phase 3 clinical trials targeting cancers with MET overexpression. However, Tivantinib ultimately was not approved as a new therapeutic, failing to show efficacy in these phase 3 clinical trials (3,4).  

Within three years of the initial publication on Tivantinib, two separate articles challenged the mechanism of action in Tivantinib-induced cytotoxicity of tumor cells (5,6). Authors for both articles showed that Tivantinib can kill both MET-addicted and nonaddicted cells with similar potency. Both articles also concluded that perturbation of microtubule dynamics, instead of MET inhibition, is likely responsible for the cytotoxicity observed with Tivantinib. Considering the failed clinical trials and uncertainties regarding the mechanism of action, one may wonder if the original pre-clinical work adequately determined if Tivantinib effectively binds and inhibits MET in cells? If Tivantinib’s cellular engagement to MET was assessed directly rather than by MET phosphorylation analysis, would a different pre-clinical recommendation have been made?

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Conjugate Like a Pro: Simplifying Antibody Labeling with On-Bead Conjugation 

Posted on by Shannon Sindermann
Antibody, On-bead conjugation

Labeled antibodies are indispensable tools in research and clinical diagnostics, used in everything from cell imaging and ELISAs to immunotherapies and ADC development. But if you’ve ever tried labeling antibodies the traditional way—purify, buffer exchange, conjugate, purify again—you know it can be tedious and time-consuming. That’s where on-bead conjugation steps in with a solution. 

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What Makes OBI-992 Different? A Closer Look at This TROP2 Antibody Drug Conjugate

Posted on by Shannon Sindermann
ADC depiction

Antibody-drug conjugates (ADCs) are an increasingly powerful class of cancer therapeutics that combine the targeted precision of monoclonal antibodies with the cytotoxic potency of small-molecule drugs. By directing chemotherapy agents specifically to tumor cells, ADCs aim to maximize antitumor activity while minimizing damage to healthy tissues. One key challenge in ADC design is selecting the right target and payload—features that define efficacy, safety and resistance. 

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Cracking the Undruggable Code: Top 10 Key Takeaways

Posted on by Sara Millevolte

For decades, the concept of “undruggable” targets has presented one of the most significant challenges in drug discovery. At our recent virtual event, Illuminating New Frontiers: Cracking the Undruggable Code, leading researchers and industry experts gathered to showcase cutting-edge technologies and fresh perspectives that are expanding the boundaries of therapeutic development. Over three engaging days, participants explored groundbreaking advances in targeting RAS signaling, leveraging protein degradation and induced proximity strategies, and exploring RNA as a therapeutic target.

Target engagement of RAF dimer inhibitor LXH254 at RAF kinases, in complex with KRAS (blue). RAF inhibitor LXH254 engages BRAF or CRAF protomers (orange), but spares ARAF (red). Unoccupied ARAF is competent trigger downstream mitogenic signaling (lightning bolts). Red cells in the background are fluorescently labeled RAS proteins, expressed in live cells.
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Targeting Dark Kinases for Non-Hormonal, Reversible Male Contraceptives

Posted on by Anna Bennett

Contraception, or birth control, is an important tool in family planning. Given the fourfold increase in population over the last century1 there is a clear need for more affordable, reversible, and safe methods of contraception. At present, the responsibility of taking contraceptives falls largely on people with female reproductive organs as there is no current method of birth control for people with male reproductive organs. The search for a non-hormonal, male birth control has been an elusive goal in the field of reproductive health.

A complex, futuristic scene within the outline of a pill.

Recently, a group of scientists from Baylor College of Medicine with contributions from Promega scientists identified a novel compound that 1) inhibits a specific kinase and 2) functions as a reversible male contraceptive. The kinase targeted in this study is the serine/threonine kinase 33 (STK33); a genetic knockout of this gene in male mice is known to cause sterility. The team published their work in Science and utilized a suite of approaches—including DNA-Encoded Libraries (DELs), crystallography, and cellular NanoBRET™ Target Engagement Kinase Assays—to discover a potent inhibitor of STK33 (CDD-2807).  The CDD-2807 inhibitor has shown promising results in inducing reversible contraception in male mice, marking a significant milestone in the development of non-hormonal contraceptive options. Let’s dive into the foundation, novel methodology, collaboration, and implications for this work.

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From Tracers to Kinetic Selectivity: Highlights from the Target Engagement in Chemical Biology Symposium

Posted on by Anna Bennett

In April 2024, Promega hosted the “Target Engagement in Chemical Biology Symposium” at the Kornberg Center, a research and development hub on Promega’s campus in Madison, Wisconsin. The goal of the symposium was to gather interdisciplinary researchers interested in the field of small molecule target engagement to foster collaboration through knowledge sharing and innovation. The symposium featured a 1.5-day agenda packed with 23 speakers, 4 workshops, poster sessions and social events. Over 130 attendees gathered to participate in the multifaceted event, with participants from different geographic regions and in different research sectors from academia to government to industry.  

People gather in a large atrium with scientific posters and table displays.
Attendees gather for the poster session in Kornberg Atrium. Photo by Anna Bennett (Promega Corporation)

The symposium highlighted the collective commitment to overcoming the challenges in drug discovery by developing more targeted and efficacious treatments, driven by a shared determination to create innovative solutions that address unmet medical needs. While we cannot share all the exciting research presented at the symposium, we are thrilled to highlight a few talks that exemplify the novel work and collaborative spirit of this research community.  

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Cancer Moonshot: Solving Tough Problems

Posted on by Michele Arduengo, PhD

At the American Association for Cancer Research meeting in April 2016, then Vice President of the United States, Joe Biden, revealed the Cancer Moonshot℠ initiative— a program with the goals of accelerating scientific discovery in cancer research, fostering greater collaboration among researchers, and improving the sharing of data (1,2). The Cancer Moonshot is part of the 21st Century Cures Act, which earmarked $1.8 billion for cancer-related initiatives over 7 years.  The National Cancer Institute (NCI) and the Cancer Moonshot program have supported over 70 programs and consortia, and more than 250 research projects.  According to the NCI, the initiative from 2017 to 2021 resulted in over 2,000 publications, 49 clinical trials and more than 30 patent filings. Additionally, the launch of trials.cancer.gov has made information about all cancer research trials accessible to anyone who needs it (3).

“We will build a future where the word ‘cancer’ loses its power.”

First Lady, Dr. Jill Biden

In February 2022, the Biden White House announced a plan to “supercharge the Cancer Moonshot as an essential effort of the Biden-Harris administration” (4).  Biden noted in his address that, in the 25 years following the Nixon administration’s enactment of the National Cancer Act in 1971, significant strides were made in understanding cancer. It is now recognized not as a single disease, but as a collection comprising over 200 distinct diseases. This period also saw the development of new therapies and enhancements in diagnosis. However, despite a reduction in the cancer death rate by more than 25% over the past 25 years, cancer continues to be the second leading cause of death in the United States [4].

The Cancer Moonshot is a holistic attempt to improve access to information, support and patient experiences, while fostering the development of new therapeutics and research approaches to studying cancer. In this article, we will focus on research, diagnostics and drug discovery developments.

Solving for Undruggable Targets

KRAS , a member of the RAS family, has long been described as “undruggable” in large part because it is a small protein with a smooth surface that does not present many places for small molecule drugs to bind. The KRAS protein acts like an off/on switch depending upon whether it has GDP or GTP bound.  KRAS mutations are associated with many cancers including colorectal cancer (CRC), non-small cell lung cancer (NSCLC), and pancreatic ductal adenocarcinoma (PDAC). The G12 position in the protein is the most commonly mutated; G12C accounts for 13% of the mutations at this site, and is the predominant substitution found in NSCLC, while G12D is prevalent in PDAC (5).

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