systems have also advanced. Especially MS-based chemoproteomic approaches are
now enabling a more complete understanding of the cellular labeling profiles of
covalent inhibitors while biotinylated or fluorescent covalent probes have also been
used as valuable tools to monitor target occupancy in cells or in vivo.
Nevertheless, we still face many challenges in the realm of covalent kinase
inhibitor discovery, especially, when aiming for clinical applications. A general
limitation of the TCI approach, especially in oncology, consists in its vulnerability
to resistance development by mutation of the nucleophilic amino acid (e.g., cysteine
to serine). This is an intrinsic liability since TCIs target non-conserved residues,
which are typically not crucial for protein integrity and function. On the other hand,
when aiming for other therapeutic areas beyond oncology where resistance mutations are less likely, an even more stringent benefit/risk assessment is required. The
latter, however, is currently complicated by a lack of reliable models to predict
toxicity and immune-mediated adverse events including idiosyncratic drug reactions, which may only become apparent in late stage clinical trials or even after longterm therapeutic application. Beside the frequently discussed issue of permanent
off-target modification, we also lack a comprehensive understanding of how reversible interactions with unintended targets contribute to the biological profiles of
reactive inhibitors, a fact that is rarely alluded to in the current literature. Similarly,
covalent modification of non-protein off-targets (e.g., DNA or RNA) by kinase TCIs
remains widely unexplored.
As mentioned in the introduction, a detailed analysis of target-binding kinetics is
still not performed by default. Thus, the contribution of the covalent binding event to
the observed overall biological effects often remains incompletely understood
[13]. Further exploration of warhead chemistry to enable the generation of inhibitors
with tailored reactivities specifically matching the requirements of the target of
interest represents another future challenge [9]. There is also an increasing awareness
of cysteine oxidation as a posttranslational modification, which may regulate kinase
function and impair covalent binding [13]. The influence of the “cysteine redoxome”
and its dynamics on the efficacy covalent inhibitors clearly merits further investigations which may have an important impact on our perspective on covalent cysteine
targeting. Finally, and despite the success of covalent approaches in the development
of chemical probes and drugs, the coverage of the kinases’ cysteinome by suitable
inhibitors is still low and several of the presented cysteine locations (see Fig. 4 and
Table 1) have not yet been targeted at all. While a thorough examination of the
protein kinases’ lysinome and tyrosinome remains to be performed, it also unclear,
how many ligandable cysteine locations, especially in inactive states or induced/
allosteric pockets have not been captured in the published analyses. The latter may
open up new avenues for covalent ligand design.
Despite the many challenges associated with covalent inhibitor development, a
bright future can be expected for protein kinase TCIs. Since many kinases are still
understudied and lack suitable chemical probes required for investigation of their
function, there is a large and untapped potential for covalent kinase inhibitor
discovery, which will inspire continuing research efforts. Moreover, a multitude of
covalent protein kinase inhibitors are currently in clinical development for
Covalent Kinase Inhibitors: An Overview
83
now enabling a more complete understanding of the cellular labeling profiles of
covalent inhibitors while biotinylated or fluorescent covalent probes have also been
used as valuable tools to monitor target occupancy in cells or in vivo.
Nevertheless, we still face many challenges in the realm of covalent kinase
inhibitor discovery, especially, when aiming for clinical applications. A general
limitation of the TCI approach, especially in oncology, consists in its vulnerability
to resistance development by mutation of the nucleophilic amino acid (e.g., cysteine
to serine). This is an intrinsic liability since TCIs target non-conserved residues,
which are typically not crucial for protein integrity and function. On the other hand,
when aiming for other therapeutic areas beyond oncology where resistance mutations are less likely, an even more stringent benefit/risk assessment is required. The
latter, however, is currently complicated by a lack of reliable models to predict
toxicity and immune-mediated adverse events including idiosyncratic drug reactions, which may only become apparent in late stage clinical trials or even after longterm therapeutic application. Beside the frequently discussed issue of permanent
off-target modification, we also lack a comprehensive understanding of how reversible interactions with unintended targets contribute to the biological profiles of
reactive inhibitors, a fact that is rarely alluded to in the current literature. Similarly,
covalent modification of non-protein off-targets (e.g., DNA or RNA) by kinase TCIs
remains widely unexplored.
As mentioned in the introduction, a detailed analysis of target-binding kinetics is
still not performed by default. Thus, the contribution of the covalent binding event to
the observed overall biological effects often remains incompletely understood
[13]. Further exploration of warhead chemistry to enable the generation of inhibitors
with tailored reactivities specifically matching the requirements of the target of
interest represents another future challenge [9]. There is also an increasing awareness
of cysteine oxidation as a posttranslational modification, which may regulate kinase
function and impair covalent binding [13]. The influence of the “cysteine redoxome”
and its dynamics on the efficacy covalent inhibitors clearly merits further investigations which may have an important impact on our perspective on covalent cysteine
targeting. Finally, and despite the success of covalent approaches in the development
of chemical probes and drugs, the coverage of the kinases’ cysteinome by suitable
inhibitors is still low and several of the presented cysteine locations (see Fig. 4 and
Table 1) have not yet been targeted at all. While a thorough examination of the
protein kinases’ lysinome and tyrosinome remains to be performed, it also unclear,
how many ligandable cysteine locations, especially in inactive states or induced/
allosteric pockets have not been captured in the published analyses. The latter may
open up new avenues for covalent ligand design.
Despite the many challenges associated with covalent inhibitor development, a
bright future can be expected for protein kinase TCIs. Since many kinases are still
understudied and lack suitable chemical probes required for investigation of their
function, there is a large and untapped potential for covalent kinase inhibitor
discovery, which will inspire continuing research efforts. Moreover, a multitude of
covalent protein kinase inhibitors are currently in clinical development for
Covalent Kinase Inhibitors: An Overview
83