Irreversible inhibitors and mechanisms of resistance -H Flashcards
Physical mechanisms of resistance in cancer
The tumours can be “protected” in areas less accessible by drugs, e.g., brain, bones, etc… Small molecules drugs often are developed to be able to enter the CNS however.
Mechanisms of resistance in signalling pathways
Bypass signalling can occur, where another RTK circumvents the inhibition of signalling by an inhibitor. Polypharmacology/combination therapy can be beneficial here.
Upstream or downstream mutations can occur, where a pathway will continue to signal, e.g., melanoma resistance to vemurafenib leading to kRas mutations or MEK/MAPK mutations.
Examples and mechanisms of mutations in the drug target leading to resistance and example of how this has been overcome (gatekeeper)
The gatekeeper residue is a small AA that enables ATP-competitive inhibitors access to a hydrophobic pocket - higher affinity for inhibitors.
Mutations that replace this AA with a bulkier AA affect the affinity of an inhibitor, but do not change ATP binding affinity.
e.g., T315I in BCR-Abl, or T790M in EGFR.
Ponatinib has been developed to be selective for both native and T315I Abl, binding with higher affinity than imatinib. It also sees reduced tyrosine kinase inhibition (could be pro or con)
Mechanism of covalent inhibitors and EGFR example
They are type V kinase inhibitors
They bind irreversibly to the kinase binding site. ATP cannot bind to the binding site, decreasing Vmax. They may also be more effective in inhibiting mutant forms of the kinase.
The acrylamide functional group of covalent inhibitors for EGFR binds covalently to the -SH group in Cys797 of the EGFR. E.g., Afatinib (second generation) or osimertinib.
Parameters that covalent inhibitor binding is dependent on
Dependent on both the affinity of the drug for the binding site, and the reactivity of the warhead (Kinact).
They do not reach equilibrium, but rather covalent binding occurs in a time dependent manner, with the endpoint dependent on [inhibitor] and [target protein].
Inhibitor IC50 is thus dependent on affinity, reactivity of warhead, and time of incubation.
The effect of reactivity of the warhead on binding kinetics
Highly reactive warheads tend to be quite non-selective - limits therapeutic use. Highly reactive warheads would bind in a 1-step mechanism, where the covalent bond occurs virtually instantaneously upon ligand-receptor binding.
For rationally designed inhibitors, they have a 2 step mechanism, where the drug is first reversibly bound (Kd), and then the warhead may react covalently with the target site (Kinact).
Role of KI and Kinact in determining potency of covalent inhibitors and how they are calculated
Kinact represents the maximum potential rate of inactivation.
KI (not same as Ki or Kd) represents the overall potency of both these binding steps. The [inhibitor] that achieves half maximal rate of inactivation.
Kinact/KI is used to identify potency on covalent inhibitors, where the higher the value, the more potent they are. This value is a rate constant, so it will be unaffected by time point of the kinetics assay.
KI and Kinact can be calculated by the Kobs of the inhibitor with the target. By looking at Kobs with different [inhibitor], mechanistic information about the inhibitor can be determined.
How do jump dilutions verify a drugs covalent mechanism of action and describe method
Preincubate a high [enzyme] and high [inhibitor]. Here the enzyme will become fully occupied.
Then dilute the mix to a lower [enzyme], while also adding a high [substrate]. If the drug is reversibly bound, the EI complexes will dissociate (according to its Koff value), and this will be identified by enzymatic activity against the substrate.
If the drug is covalent, enzymatic activity should not be seen.
Potential of future therapeutic drugs for kinases
PROTACs (proteolysis targeting chimeras) are bivalent molecules that contain a kinase inhibitor and a degrader.
The degrader binds to ubiquitin E3 ligase. As such, upon the PROTAC binding to the kinase, it will bring the E3 ligase into its proximity. This would initiate and enhance ubiquitination and proteosomal degradation of the kinase, while simultaneously inhibiting its action through a conventional MOA.
