Kinases Flashcards

Question

Rational design of type II inhibitors **

Answer 1

Type II kinase inhibitors bind kinase in their inactive, DFG-out conformation, at a hydrophobic pocket that is less conserved than the ATP binding site. We can design these by starting with known type I inhibitors. Select a type I scaffold: Choose a scaffold with known activity against the desired kinase and suitable for attaching a linker to the type II hydrophobic tail. Attach a type II tail: Append a moiety containing the hydrogen bond donor-acceptor pair and the hydrophobic motif to the type I scaffold via the linker. Verify binding mode: Dock the designed molecule into a DFG-out kinase structure to ensure the type I head and type II tail fit the ATP and allosteric pockets with appropriate interactions.

Answer 2

Resistance can occur through the protection of tumours in “sanctuary” sites less accessible to drugs, such as the brain metastases, which require CNS penetration of the drug for activity. However, small molecule kinase inhibitors are often developed to be blood brain barrier penetrant (e.g. highly lipophilic), and this is more of an issue for biologics like antibodies. Cancer cells can acquire additional mutations that generate resistance to the pharmacological effect of the drug. These seed populations expand to become the predominant form in the tumour. Cancers can bypass signalling pathways that have been inhibited, so another kinase circumvents the inhibition of the first. In NSCLC, EGF receptor inhibition by Gefitinib is often circumvented by overexpression of another RTK that activates the MAPK pathway, MET. Upstream/downstream mutations can also activate the pathway above/below the drug target. Vemurafenib inhibits the B-Raf serine/threonine kinase. Resistant melanoma cells acquire activating RTK mutations in the upstream Ras kinase (G12V) or the downstream MEK or MAPK. This is why polypharmacology can be beneficial. In the case above, combination therapy, for example with a B-Raf inhibitor and a MEK or MAPK inhibitor would overcome the resistance.

Answer 3

A variety of mutations may be acquired in the kinase catalytic domain that selectively reduce binding of the inhibitor but not that of ATP. The most important mutation site for many different kinases is the gatekeeper residue. This is an amino acid with a small side chain, such as threonine, that allows ATP-competitive inhibitor access to a small hydrophobic binding pocket to generate high affinity inhibition (relevant to type II inhibitors). Mutations replace this with a bulky side chain, preventing inhibitor binding. - Thr315Ile (T315I) mutation in BCR-Abl causes resistance to imatinib. - Thr790Met (T790M) mutation in EGFR causes resistance to gefitinib. This led to the development of a new generation of BCR-Abl inhibitors. Ponatinib binds both native and T315I Abl with a higher affinity than imatinib. It also shows a reduction in selectivity, which could be a pro or con.

Answer 4

Type V covalent inhibitors bind via an irreversible mechanism, in which they covalently modify the kinase binding site. After covalent modification, ATP cannot compete for the kinase binding site and surmount the inhibition. There is an effective reduction of kinase activity (Vmax) in cells with high [ATP]. Covalent mechanisms may also lead to more effective inhibition of resistance mutant kinases, for example after gatekeeper mutation. There is a risk that the covalent mechanism could enhance on/off target side effects due to increased reactivity. If covalency is non-selective, the drug becomes a poison. Covalent EGFR inhibitors, like afatinib, have an acrylamide functional group that reacts with the Cys797 side chain (-SH) of the EGFR to form the covalent bond. Third-generation drugs, like rociletinib, are covalent inhibitors that deal with the T790M gatekeeper mutation acquired in resistance.

Answer 5

IC50 cannot be used as a measure of potency for covalent inhibitors, as these are non-surmountable and so equilibrium is NOT reached. The time of incubation will therefore affect the IC50 value, so this will not be constant. Inhibitor potency depends on a combination of affinity (KD) and the reactivity of the warhead (Kinact). Warheads are electrophilic groups in covalent kinase inhibitors that form the covalent bond. In competition biochemical assays, product formation over time decreases with increasing concentrations of inhibitor, as the enzyme becomes inactivated. Measuring inhibitor IC50 curves over time shows a left shift, as the inhibitor becomes more potent over time. There is a challenge as we can have low and high affinity covalent inhibitors, but if enough time is given these will appear to have the same IC50, which will be half the kinase concentration.

Answer 6

Highly-reactive inhibitors follow a one-step mechanism, immediately forming a covalent bond. High reactivity however limits selectivity, so these are often not pharmacologically useful. Most inhibitors follow a two-step mechanism. Step 1 – reversible binding, governed by the association and dissociation rate constants, kon and koff, and so the inhibitor KD (KD = koff/kon). Step 2 – irreversible covalent reaction once bound - Kinact represents the maximum potential rate of kinase inactivation. This is common for rationally designed inhibitors with high affinity for the target site, and titrated reactivity through choice of warhead → we design inhibitors with high affinity and then titrate activity down to obtain high selectivity. KI represents the overall potency of the binding steps. It is the concentration of inhibitor that achieves the half maximal rate of inactivation. KI = (koff + kinact) / kon It is not the same as KD for the inhibitor, but KI ~ KD where koff >>> kinact kinact / KI is a useful single parameter to determine the rank order of covalent inhibitors in SAR. Higher potency/more reactive inhibitors have a higher ratio. kinact / KI is a rate constant and is derived from the kinetic time course of the data, so its measurement for individual inhibitors is not influenced by the choice of one assay time point. kinact and KI can be estimated from kinetic binding or biochemical studies measuring the observed rate of inhibitor association to the target, kobs. kobs = kinact[inhibitor] / KI + [inhibitor] Analysis of how kobs varies with inhibitor concentration can also provide mechanistic information about one step or two step covalent models.

Answer 7

The jump dilution method can be used in biochemical assays to verify a covalent mechanism of inhibition. 1. Preincubate a high concentration of the enzyme and inhibitor. During this step, the enzyme preparation becomes fully occupied by inhibitor molecules. 2. Dilute the mix to a (much lower) working concentration of enzyme (100-1000 fold). If the inhibitor is reversible, EI complexes gradually dissociate at a rate related to the inhibitor koff. Covalent inhibitors do not dissociate. 3. Measure activity over time in presence of high [substrate]. If product is not formed, the enzyme is covalent. If product is detected, binding must be reversible. Changing experimental conditions can help us understand the reactivity of the covalent warhead - e.g. What pretreatment time is required to achieve irreversibility?

Answer 8

Another possible avenue to achieve long lasting non-surmountable inhibition is degradation of the target kinase protein. PROTACs (Proteolysis targeting chimeras) are bivalent molecules that link a kinase inhibitor to a degrader that binds a ubiquitin E3 ligase, hijacking the ubiquitin-proteosome system. Binding of the PROTAC to the target kinase also brings the E3 ligase into its proximity through the degrader arm of the molecule. - There is a region able to fit into the kinase ATP site and another able to bind E3 ligase, connected by a linker. The aim is to enhance ubiquitination and proteasomal degradation of the kinase protein, as well as pharmacological inhibition. PROTAC technology has shown promise in preclinical studies targeting various protein kinases, including Akt (PKB), SGK3, and Abl. Advantages: - Increased selectivity by exploiting specific E3 ligases. - Potentially circumventing resistance mechanisms such as gatekeeper mutations as they target the protein itself rather than its catalytic activity. Challenges: - Cellular Permeability: PROTACs are larger than small-molecule inhibitors. - Maintaining the stability of the linker between the targeting and ligase-binding components is crucial for PROTAC function. - Unintended degradation of non-target proteins due to promiscuous E3 ligase binding can lead to toxicity.

Answer 9

Ligand-first approach: This approach involves incorporating a reactive electrophilic group into an existing ligand that already binds reversibly to the target protein. Electrophile-first approach: This method involves identifying covalent ligands directly from libraries of electrophilic compounds. This strategy has gained traction in recent years, particularly with the development of high-throughput screening platforms and chemoproteomics.

Kinases Flashcards

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