Conformational Effects of Clinically Approved BTK Inhibitors and Mechanisms of BTK Resistance Mutations in Chronic Lymphocytic Leukemia

מושגי ליבה

This research paper investigates the distinct conformational changes induced by clinically approved BTK inhibitors on full-length BTK and elucidates the mechanisms of action of BTK resistance mutations, particularly T474I and L528W, in chronic lymphocytic leukemia.

תקציר

• Bibliographic Information: Not provided in the content.
• Research Objective: This study aims to characterize the unique conformational changes induced by five clinically approved BTK inhibitors (Ibrutinib, Acalabrutinib, Zanubrutinib, Tirabrutinib, and Pirtobrutinib) on full-length BTK and to investigate the mechanisms of action of two common BTK resistance mutations, T474I and L528W, observed in chronic lymphocytic leukemia (CLL) patients.
• Methodology: The researchers employed a multidisciplinary approach, including nuclear magnetic resonance (NMR) spectroscopy, hydrogen-deuterium exchange mass spectrometry (HDX-MS), in vitro kinase assays, and western blotting, to analyze the structural and functional consequences of BTK inhibitor binding and resistance mutations.
• Key Findings:
  • Each BTK inhibitor induced distinct conformational changes in full-length BTK, with Acalabrutinib, Zanubrutinib, and Tirabrutinib destabilizing the autoinhibited conformation, while Pirtobrutinib stabilized it.
  • The BTK T474I mutation reduced inhibitor binding affinity, particularly for Zanubrutinib, Tirabrutinib, and Pirtobrutinib, potentially explaining its role in resistance.
  • The catalytically inactive BTK L528W mutant activated the SRC kinase HCK in a proline-rich region-dependent manner, suggesting an alternative signaling pathway for CLL cell survival.
  • The BTK L528W mutation also increased BTK protein stability, potentially contributing to its resistance mechanism.
• Main Conclusions: The study highlights the diverse effects of clinically approved BTK inhibitors on BTK conformation and provides insights into the mechanisms of action of BTK resistance mutations. These findings have implications for understanding the differential efficacy of BTK inhibitors and for developing strategies to overcome resistance in CLL.
• Significance: This research advances the understanding of BTK inhibitor resistance mechanisms in CLL, which is crucial for improving treatment strategies and developing next-generation inhibitors that can overcome resistance.
• Limitations and Future Research: The study primarily focused on two specific BTK resistance mutations. Further investigation into other resistance mutations and their impact on BTK structure and function is warranted. Additionally, in vivo studies are needed to validate the clinical relevance of the findings.

סטטיסטיקה

In Zanubrutinib-treated patients, the L528W mutation was often present together with the C481S mutation (on different alleles), and with the L528W mutation present at a higher allelic frequency compared to C481S.
90% or more of CLL patients treated with Ibrutinib and Acalabrutinib develop mutations in BTK C481.
Substitution of C481 with serine is the most common resistance mutation found in these patients.

ציטוטים

תובנות מפתח מזוקקות מ:

Impact of the clinically approved BTK inhibitors on the conformation of full-length BTK and analysis of the development of BTK resistance mutations in chronic lymphocytic leukemia

by Joseph,R. E.... ב- www.biorxiv.org 12-18-2023

https://www.biorxiv.org/content/10.1101/2023.12.18.572223v2

שאלות מעמיקות

How could the findings of this study be applied to the development of novel BTK inhibitors or combination therapies for CLL?

This study provides several crucial insights that could be leveraged for the development of novel BTK inhibitors and combination therapies for CLL:
1. Targeting the BTK autoinhibited conformation:

Developing inhibitors that mimic Pirtobrutinib's effect: The study identifies Pirtobrutinib as a unique inhibitor that stabilizes the autoinhibited conformation of BTK. This suggests a novel strategy for drug design, focusing on developing molecules that mimic this effect. Such inhibitors could potentially offer higher efficacy and a distinct resistance profile compared to existing drugs.
Exploiting the autoinhibited state for combination therapies: Stabilizing the autoinhibited conformation could also enhance the efficacy of other drugs. Combining a Pirtobrutinib-like inhibitor with another targeted therapy could synergistically inhibit BTK signaling and potentially overcome or delay resistance.
2. Overcoming resistance mutations:

Designing inhibitors that bypass resistance mutations: The study elucidates the structural basis for resistance mutations like T474I and L528W. This knowledge can guide the design of next-generation BTK inhibitors with modified structures that can effectively bind to and inhibit these mutant forms of BTK.
Developing drugs targeting alternative binding sites: Understanding the conformational changes induced by different inhibitors can help identify novel allosteric binding sites on BTK. Targeting these sites could circumvent resistance arising from mutations in the active site.
3. Exploiting the BTK-HCK interaction:

Disrupting the BTK-HCK interaction: The study reveals the activation of HCK by the BTK L528W mutant through its proline-rich region. Developing inhibitors that specifically block this interaction could be a promising strategy to overcome resistance mediated by this mutation.
Combination therapy targeting both BTK and HCK: Simultaneously inhibiting both BTK and HCK could be an effective approach to shut down BCR signaling in resistant CLL cells. This could involve combining existing BTK inhibitors with HCK inhibitors or developing dual inhibitors targeting both kinases.
4. Considering inhibitor half-life in drug design:

Optimizing inhibitor half-life to minimize C481S mutation: The study highlights the correlation between inhibitor half-life and the prevalence of the C481S mutation. This finding emphasizes the importance of considering pharmacokinetic properties like half-life during drug development. Designing inhibitors with optimal half-lives could potentially minimize the emergence of specific resistance mutations.
By integrating these insights into drug discovery efforts, researchers can develop more effective and durable treatment strategies for CLL patients, including those who develop resistance to current BTK inhibitors.

Could the BTK L528W mutation's activation of HCK be targeted therapeutically to overcome resistance in CLL?

Yes, the BTK L528W mutation's activation of HCK presents a promising therapeutic target to overcome resistance in CLL. Here's why and how:
Why it's a viable target:

Essential role of HCK in BTK L528W-mediated signaling: The study demonstrates that despite being catalytically inactive, the BTK L528W mutant can still propagate B-cell receptor (BCR) signaling by activating HCK. This highlights HCK as a crucial downstream effector in maintaining the survival and proliferation of CLL cells harboring this resistance mutation.
Dependence on proline-rich region interaction: The activation of HCK by BTK L528W is dependent on the interaction between the proline-rich region of BTK and the SH3 domain of HCK. This interaction represents a specific and targetable vulnerability in the resistance mechanism.
How it can be targeted:

Developing inhibitors of the BTK-HCK interaction: Small molecule inhibitors or peptidomimetics could be designed to specifically block the interaction between the proline-rich region of BTK and the SH3 domain of HCK. This would prevent HCK activation and disrupt downstream signaling, even in the presence of the BTK L528W mutation.
Utilizing existing HCK inhibitors: Several HCK inhibitors are currently under investigation for various cancers. Repurposing these inhibitors, either alone or in combination with BTK inhibitors, could be a rapid and potentially effective strategy to target HCK activity in BTK L528W-resistant CLL.
Exploring PROTAC technology: Proteolysis targeting chimeras (PROTACs) are a novel class of drugs that induce targeted protein degradation. PROTACs could be designed to specifically degrade HCK in BTK L528W-resistant CLL cells, providing a sustained and potentially more durable therapeutic effect.
Challenges and considerations:

Specificity and off-target effects: HCK plays a role in other cellular processes, so inhibiting it could lead to unintended side effects. Developing highly specific inhibitors or targeting strategies that minimize off-target effects will be crucial.
Clinical development and validation: Rigorous preclinical and clinical studies will be necessary to evaluate the safety, efficacy, and optimal dosing strategies for any therapeutic approach targeting HCK in this context.
Despite these challenges, targeting the BTK L528W-HCK interaction represents a promising avenue for developing novel therapies to overcome resistance and improve outcomes for CLL patients.

What are the broader implications of understanding protein conformational changes induced by drug binding for drug discovery and development in other diseases?

Understanding the intricate relationship between protein conformational changes and drug binding has profound implications for drug discovery and development across a wide range of diseases. This study's findings, particularly in the context of BTK inhibitors, highlight several key takeaways with broad applicability:
1. Moving beyond static drug design:

Dynamic interplay between drugs and targets: This study underscores the dynamic nature of proteins and how drug binding can induce significant conformational changes, influencing protein function. This emphasizes the limitations of traditional drug design approaches that rely solely on static protein structures.
Importance of considering conformational ensembles: Future drug discovery efforts should consider the dynamic ensemble of protein conformations rather than focusing solely on a single static structure. This approach can lead to the identification of novel binding pockets and allosteric sites that are not apparent from static structures.
2. Understanding and predicting drug resistance:

Conformational changes as a driver of resistance: This study demonstrates how mutations can alter protein conformation and subsequently affect drug binding, leading to resistance. Understanding these conformational shifts can help predict potential resistance mutations and guide the development of next-generation drugs that are less susceptible to resistance.
Developing drugs targeting resistant conformations: By characterizing the conformational changes associated with resistance mutations, researchers can design drugs that specifically target these altered protein states, potentially overcoming or circumventing resistance mechanisms.
3. Exploiting allostery for drug development:

Targeting allosteric sites for enhanced specificity and efficacy: This study highlights the potential of allosteric drug binding to modulate protein function. Allosteric inhibitors, like Pirtobrutinib, can offer advantages over traditional active site inhibitors, including higher specificity and potentially fewer off-target effects.
Developing allosteric activators for therapeutic benefit: Understanding allosteric mechanisms can also facilitate the development of drugs that activate protein function, which could be beneficial for diseases characterized by protein deficiencies or loss of function.
4. Expanding the toolkit for drug discovery:

Integrating biophysical techniques for comprehensive analysis: This study utilizes a combination of techniques, including HDX-MS and NMR, to characterize protein conformational changes upon drug binding. Integrating such biophysical methods into drug discovery workflows can provide a more comprehensive understanding of drug-target interactions and facilitate the development of more effective therapies.
Leveraging computational approaches for drug design: Advances in computational modeling and simulation techniques, coupled with experimental data on protein dynamics, can enable the design of drugs that specifically target desired protein conformations.
In conclusion, understanding the dynamic interplay between drug binding and protein conformational changes is crucial for advancing drug discovery and development across various diseases. By embracing this dynamic perspective and leveraging evolving technologies, researchers can develop safer, more effective, and durable therapies for a wide range of human ailments.