irreversible inhibitors and mechanism of resistance

Question 1

list the mechanisms of kinase inhibitor resistance

Answer 1

1. protecting tumors in sites less accessible to drug. ex: brain metastases. (hard for drugs to get into cns) However, small mollecule kinase inhibitors are designed to be bbb penetrative. this is more an issue for biologic kinase inhibitors.
2. mutations that generate resistance. little mutated populations expand and become the prominent form of the tumor
3. bypass signalling (another rtk circumvents inhibition of first rtk.) ex: EGFR activating MAPK blocked by 1st gen gefitinib. MET is overexpressed in NSCLC. this also activates MAPk
4. upstream/downstream mutations in pathway activating targets below or above.
   ex: Vemurafenib targets B-raf v600E. RAS mutation G12V upstream increases drive to overcome raf inhibition.

Question 2

how can mechanisms of kinase inhibitor resistance be overcome

Answer 2

1. polypharmacology (less selective RTK inhibition)
2. Combination therapy. ex: B-raf and MEK inhibitor used together

Question 3

How does inhibitor potency differ for type 5 kinase inhibitors

Answer 3

IC50 depends on
1. affinity (kd)
2. reactivity of warhead (electrophilic functional group) ex: acrylamide
3. Time of incubation
İnhibition is not surmountable by adding more substrate of ATP
equilibrium wont be reached, covalent reactions are time dependent and endpoint only gets limited by inhibitor concentration and target protein levels. (inhibitor will seem to become more potent with longer time)

Question 3

what is the gatekeeper residue and what are implications of its mutation

Answer 3

AA near the ATP binding pocket of kinases.
Normally this AA has a small side chain which allows ATP competitive inhibitors (type 1 and 2) to bind
Mutations that make this side chain bulky are undesirable as it prevents inhibitor binding. (mutations making it smaller are beneficial)
ex:
T315I mutation is BCR-abl generates resistance to imatinib
T790M EGFR generates resistance to gefitinib.

new generation BCR-abl inhibitors Ponatinib binds to both native and T315I Abl (higher affinity for mutated)

Question 3

what are type 5 kinase inhibitors

Answer 3

they are irreversible inhibitors as they bind covalently to modify the binding site so that ATP cannot compete for the binding site.
covalent mechanisms can lead to more effective inhibition of kinases that have gatekeeper mutations.
risks: enhanced side effects
ex: Neratinib, afatinib, dacomitinib. they have an acrylamide funtional group that forms covalent bonds with Cys residues. (second gen)

third gen: osimetinib, rociletinib (T790M gatekeeper mutation selective but target native too)

Question 4

describe the two step mechanism of covalent inhibitors

Answer 4

E + I = (reversible Kon vs Koff) EI = (covalently bound Kinact) E-I

step 1 is governed by ass. diss rate constants kon and koff.
KD= koff/kon

step 2 is irreversible. Kinact represents maximum potential rate of inactivation

KI represents overall potency
KI= (koff + Kinact) / Kon

if Kinact/KI is higher then inhibitor has higher potency
it is a rate constant. (unlike IC50 focused on single timepoints)
Ki and Kinact can be estimated from Kobs (observed rate of inhibitor association)

Question 5

what is jump dilution

Answer 5

biochemical assay used to verify if mechanism of inhibition is covalent.

you preincubate high concentrations of enzyme and inhibitor together. (so that enzyme becomes fully occupied with inhibitors.)
then dilute to a much lower working enzyme concentration and measure enzyme activity over time in presence of substrate. (If irreversible gradually inhibitors dissociate and substrate produces product.)

Question 6

What are PROTACS

Answer 6

Proteolysis targeting chimeras
another prospective way to achieve long lasting non surmountable inhibition.
they link kinase inhibitors to degrader. (bifunctional substrate-Linker-degrarder)
degrader binds to ubiquitin E3 ligase
enahcnes ubiquination and proteosomal degredation of kinases.