Enhancing Reversible Covalent Drug Design

Kinetic aspects of Reversible Covalent Drug Design

Irreversible covalent drugs offer numerous advantages over reversible non-covalent inhibitors. Despite these benefits, irreversible covalent drugs encounter challenges such as off-target toxicities and immunogenic risks.1 Introducing reversibility can mitigate these issues. As reversible covalent inhibitors are not permanently bound, they can detach from unintended proteins, reducing the risk of immune system activation and off-target toxicity.

Figure 1. Advantages and challenges of covalent drugs.

Kinetic aspects of Reversible Covalent Drug Design

By tuning the reactivity of the warhead (k3) and the residence time, reversible covalent inhibitors can maintain the benefits of irreversible covalent inhibitors while limiting off-target toxicity. This involves firstly modifying the warhead’s reactivity and structurally stabilizing the reversible covalent adduct to prolong its effectiveness.2 Secondly, optimizing the residence time with both on- and off-target proteins is crucial for minimizing toxicity. A high dissociation rate from secondary targets allows for quick reversal of any accidental covalent bonding.

Figure 2. Mechanism for reversible covalent inhibition.

Analysis of the Reversible Covalent BTK Inhibitor Rilzabrutinib using COVALfinder® and KINETICfinder®

Bruton’s tyrosine kinase (BTK), expressed in B cells and mast cells, plays a critical role in multiple immune-mediated disease processes. Rilzabrutinib is a very potent reversible BTK inhibitor that engages a noncatalytic cysteine (Cys481) present in BTK in a covalent manner. Currently, Rilzabrutinib is undergoing Phase III trials for adults and adolescents with persistent or chronic immune thrombocytopenia and has been studied in numerous Phase II trials for other immune disorders3,4

Analyzing both reactivity and reversibility is crucial for the optimization of Reversible Covalent Drugs

COVALfinder® and KINETICfinder® platforms have been used in combination to provide an in-depth understanding of the binding mechanism of Rilzabrutinib with its primary target BTK and secondary targets HER4 and ITK. Our results confirm that BTK-Rilzabrutinib, HER4-Rilzabrutinib and ITK-Rilzabrutinib complex formation each take place in reversible sequential events: such that Rilzabrutinib binding to BTK, HER4 and ITK is the initial event followed by covalent bond formation with the non-catalytic cysteine of BTK, HER4 and ITK as the second step (Fig. 3-4).

Our findings also reveal that Rilzabrutinib rapidly forms a covalent bond with the non-catalytic cysteine of BTK, HER4 and ITK. The half-life for this process is similar between the main and secondary targets (1.5, 1.7 and 2.3 minutes, respectively). Moreover, the covalent bond between Rilzabrutinib and BTK, HER4 and ITK is completely reversible (residence time of 812, 225 and 44 minutes respectively).

The high affinity of Rilzabrutinib to BTK (Kd*: 0.14 nM) is a result of the extraordinary stability of the BTK-Rilzabrutinib complex (koff: 2.0×10-5 s-1). This enables Rilzabrutinib to mimic the long-lasting activity of an irreversible covalent inhibitor, with its in vivo dissociation rate controlled by BTK degradation and resynthesis.

Figure 3. Characterization of the kinetics of BTK inhibition by the reversible covalent drug Rilzabrutinib. (A) Progress curve of BTK incubated with increasing compound concentrations. (B) Dependence of kobs on Rilzabrutinib concentration. (C) Dose-response curves over time. (D) IC50 values over time.
Tabla de datos
Binding step Covalent bond formation step
Target k1 (M−1s−1) k2 (s−1) Kd (nM) k3 (s−1) k4 (s−1) Kd* (nM) koff (s−1) Residence time (min)
BTK 3.30×105 1.49×10−2 45 1.12×10−2 3.60×10−5 0.15 2.05×10−5 812
HER4 2.19×105 8.24×10−3 38 9.84×10−3 1.64×10−4 0.62 7.42×10−5 225
ITK 5.61×104 2.95×10−2 527 7.15×10−3 4.93×10−4 34 3.92×10−4 43

Table 1. Kinetic constants, residence time and affinity values of Rilzabrutinib for BTK, HER4 and ITK measured by COVALfinder® and KINETICfinder®

Our results support those of Bradshaw et al. who found that, in rats, BTK inhibition remained around 57% after 840 minutes (14 hours), even though the serum concentration of Rilzabrutinib dropped to less than 3 ng/mL from nearly 500 ng/mL after 1 hour at a 40 mg/kg dose. Despite being cleared from circulation, Rilzabrutinib showed significant target engagement 14 hours after oral dosing, reflecting its slow dissociation from BTK in vivo.2

Kinetic selectivity of Rilzabrutinib maximizes its therapeutic window

Rilzabrutinib has outstanding kinome-wide selectivity, avoiding all kinases that lack the conserved cysteine as well as many physiologically important kinases that have this cysteine, including EGFR, HER2, JAK3 and MKK7.2 This exceptional selectivity is achieved through two distinct mechanisms utilized by reversible covalent drugs:

  • Forming a bond with a reactive amino acid residue.
  • Tuning inhibitor residence time against on- and off-targets.
  • The residence time of Senexin C binding to CDK8 or CDK19 is 2−3 fold longer than that of Senexin B, suggesting a potential mechanism for the increased efficacy of Senexin C over Senexin B.
  • Cell-based drug wash-off experiments indicated that CDK8/19 inhibition was far more durable after treatment with Senexin C relative to Senexin B, which is in agreement with the longer residence time of Senexin C.




Figure 4. Kinetic curves of Rilzabrutinib for BTK, HER4 and ITK obtained with KINETICfinder®. KINETICfinder® assays were used to determine the association rate constant (k1) of the initial step

An important benefit of fine-tuning residence time is the ability to achieve high sustained occupancy of the target without the need for extended exposure to the compound, whilst also limiting off-target toxicity. In a phase I study, Rilzabrutinib demonstrated a rapid elimination half-life (t1/2) of 3.20 hours5. Despite this rapid clearance, Rilzabrutinib’s prolonged residence time on BTK (14 hours) guarantees sustained target inhibition6.

The shorter residence times on ITK (43 minutes) and HER4 (3.75 hours) mean that any initial off-target binding is quickly reversible. This kinetic selectivity, combined with the rapid elimination, helps in reducing the duration and impact of off-target effects, improving the drug’s safety profile. Consequently, Rilzabrutinib’s therapeutic window is maximized. The chance of adverse side effects is minimized, and tolerability improves while efficacy is maintained over an extended period of time.

References

  1. Faridoon et al. (2023) An update on the discovery and development of reversible covalent inhibitors. Med Chem Res 32(6):1039-1062.
  2. Bradshaw M.J. et al. (2015) Prolonged and tunable residence time using reversible covalent kinase inhibitors. Nat Chem Biol. 11(7):525-31.
  3. Pate D. et al (2024) Reversible Covalent Inhibition─Desired Covalent Adduct Formation by Mass Action. ACS Chem Biol.
  4. Langrish C.L. et al. (2021) Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease. J Immunol 1;206(7):1454-1468.
  5. Ucpinar S. et al. (2023) Rilzabrutinib, a reversible covalent Bruton’s tyrosine kinase inhibitor: Absorption, metabolism, excretion, and absolute bioavailability in healthy participants. Clin Transl Sci 16(7):1210-1219.
  6. Smith PF. et al. (2017) A phase I trial of PRN1008, a novel reversible covalent inhibitor of Bruton’s tyrosine kinase, in healthy volunteers. Br J Clin Pharmacol 83:2367-2376.