Kinases

Question 1

Kinase selectivity

Answer 1

The catalytic actions and targets of individual kinases are highly selective.
They target amino acid side chain hydroxyls OH (Ser, Thr or Tyr).
- Serine / Threonine kinases (e.g. PKA, PKC, Akt/PKB)
- Tyrosine kinases (e.g. EGF receptor, VEGF receptor, src)
- Dual specificity (Ser / Tyr) kinases (e.g. MEK)
Look at consensus sequences for the target phosphorylated residue, i.e. the context in which the amino acids are found.
Subcellular localisation and access to the target proteins also play a role.

Question 2

GPCR regulation of kinases

Answer 2

Protein kinases A, G, and C are in part regulated by G proteins (GPCRs).
PKA is activated by Gs cAMP signalling
PKG is activated by cGMP signalling
PKC is activated by DAG from Gq signalling

Question 3

RTKs

Answer 3

Receptor tyrosine kinases are common examples within the catalytic receptor superfamily.
Receptor tyrosine kinases are involved in cell fate and metabolism decisions, including cell proliferation, survival and differentiation.
- NGF/TrkA responsible for neurite outgrowth and differentiation.
- VEGF receptors responsible for angiogenesis, the growth of new blood vessels. This is important in tumour development as it allows the tumour to gain access to oxygen and nutrients and to metastasize.
Following the binding of a growth factor, the receptor dimerises. The tyrosine kinase of one receptor phosphorylates its partner, and vice versa.
Signalling proteins are recruited to the phosphorylated RTK. These proteins contain an SH2 domain which recognises the phosphorylated Tyr and surrounding amino acids in the receptor, providing specificity. The SH3 domain is bound by proline-rich regions of other signalling proteins.

Question 4

RTKs - MAP kinase pathway

Answer 4

Involved in gene expression, protein translation and cell proliferation.
Adaptor proteins Grb2 and SOS link the phosphorylated EGFR to the activation of the small G protein Ras. SOS is a guanine nucleotide exchange factor.
Ras-GTP activates a sequential protein kinase cascade: activates Raf kinase, which then phosphorylates and activates MEK (aka MAPKK) and then MAPK (aka ERK).
MAPK leads to gene transcription and cell proliferation.
MAPK phosphatase switches off this cascade.
Kinase subtypes and isoforms exist for all the components to tailor responses. There are also molecular differences in how activation occurs and the types of signalling pathways activated.

Question 5

RTKs - PI3K pathway

Answer 5

An adaptor protein activates PI3K.
PI3K converts PIP2 to PIP3.
PIP3 activates phosphoinositide-dependent kinase (PDK1) to AKT (PKB) which then phosphorylates other proteins.
The off-switch is phosphatase and tensin homolog protein (PTEN), which converts PIP3 back to PIP2.

Question 6

RTKs - PLC𝛾 pathway

Answer 6

PLC-𝛾 can directly interact with phosphorylated RTK, leading to increased intracellular [Ca2+] .
PIP2 is phosphorylated to IP3 and DAG.
IP3 causes the release of Ca2+ from the endoplasmic reticulum and DAG activates PKC.
The off-switch is inositol monophosphatase (IMPase), which breaks down IP3.

Question 7

Non-receptor tyrosine kinases

Answer 7

Non-receptor tyrosine kinases are intracellular and do not have a ligand binding domain → not activated by a single messenger
These have key signalling nodes for many signalling pathways in cells, such as SRC, FYN, LCK and JAK

Question 8

Non-receptor tyrosine kinases - JAK/STAT pathway

Answer 8

The JAK/STAT pathway is involved in immune cytokine signalling, such as with IL-6
IL-6 is a pro-inflammatory cytokine released from macrophages.
It activates a complex of the IL-6 receptor and gp-130 co-receptors (two).
JAK is a cytoplasmic tyrosine kinase recruited to the active receptor. It phosphorylates gp-130.
This leads to recruitment and phosphorylation of the transcription factor STAT. STAT then dimerises and translocates to the nucleus, where it regulates pro-inflammatory gene expression.
The response is tailored for different signalling pathways by the existence of multiple receptor/JAK/STAT isoforms and cell type dependent expression and regulation.

Question 9

Targeting kinases

Answer 9

We can target the kinase catalytic domain with small molecules. This can be at the ATP binding site - e.g. Gefitinib at the kinase domain of the EGFR for NSCLC.
Limitations: ATP site is highly conserved, inhibitors compete with high intracellular [ATP], cell permeability is a barrier, resistance can develop.
We can target the growth factor agonist with antibodies - e.g. Bevacizumab is an anti-VEGFA monoclonal antibody, preventing receptor interaction in various cancers.
We can similarly target the extracellular RTK domain - e.g. Cetuximab is an anti-EGFR antibody, acting as an antagonist of EGF binding in NSCLC treatment. Ramucirumab is a fully-human IgG1 monoclonal antibody against the extracellular domain of VEGFR2, used in metastatic NSCLC and metastatic colorectal cancer. Trastuzumab (Herceptin) binds HER2 in the treatment of breast cancer.
Antibody therapeutics can also be conjugated with cytotoxic drugs for targeted delivery, avoiding system toxicity - e.g. Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that combines trastuzumab with the potent cytotoxic agent, emtansine.
Challenges: selectivity between closely related isoforms, delivery and tumour access, Immunogenicity, resistance.

Question 10

Cancer

Answer 10

Overexpressed or mutated kinase pathways are involved in many hallmarks of cancer.
- Proliferation - EGFR
- Immunosuppression - TAM kinases
- Metastasis - cMet (RTK involved in cell motility)
- Angiogenesis - VEGFR
Cancer is a very heterogeneous disease - the same cancer can be driven by different factors in different individuals.
Tumour diagnosis using genotyping and phenotyping is carried out to identify personalised treatments - e.g. Trastuzumab is only used in HER2+ breast cancer.
In general, clinical treatment with kinase inhibitors is most effective when cancers are identified at an early stage .

Question 11

Approved kinase inhibitors for cancer

Answer 11

Chronic myeloid leukaemia (CML) is a cancer of white blood cells, involving unregulated growth of myeloid cells in the bone marrow and proliferation of granulocytes such as neutrophils.
It’s associated with the Philadelphia chromosome resulting from chromosomal translocation. This creates a tumour associated fusion gene and overexpressed kinase protein BCR-Abl. Abl is a tyrosine kinase and BCR makes it constitutively active, leading to increased proliferation.
There’s oncogenic addiction, so tumour survival depends on one pathway.
BCR-Abl can be inhibited by Imatinib, which binds to the tyrosine kinase domain and stabilises the protein in its closed, inactive conformation. It’s a type 2 inhibitor, accessing a hydrophobic region unique to the fusion protein, which improves its specificity.
Resistance mechanisms can develop such as increased drug efflux/decreased uptake and kinase point mutations.
US death rate halved from 0.9 to 0.4 per 100,000 patients following imatinib introduction.

Question 12

Non-cancer indications

Answer 12

Infliximab is a monoclonal antibody that binds cytokines like TNF-α, used for inflammation and immune diseases, such as IBD and Crohn’s disease.
Tofacitinib is a small molecule that selectively targets JAK kinase, offering more general inhibition of immune cytokine signalling. It may be more efficacious and it can also be taken orally rather than via injection, but there is a higher risk of side effects like infection due to immunosuppression.
Fasudil targets Rho kinase in vascular SM in the treatment of cerebral vasospasm, acting as a vasodilator.
Neflamapimod is a p38 MAPK inhibitor that could be used in Alzheimer’s disease. p38 MAPK is highly expressed in microglia and brain cells in AD, and it causes neuroinflammation and tau localisation and affects neuronal plasticity.
Leucine-rich repeat kinase 2 (LRRK2) mutations could be targeted Parkinson’s disease. LRRK2 is a serine/threonine kinase that plays a diverse role in cellular function and engages in protein-protein interactions that influence its cellular localization and function. Mutations are associated with PD as they cause increased kinase activity and altered protein interactions.

Question 13

Kinase structure

Answer 13

The regulatory domain is very diverse. In RTKs, these include ligand binding / membrane spanning and juxtamembrane sequences.
The catalytic domain that binds ATP and carries our phosphorylation is highly conserved.

Question 14

The catalytic domain

Answer 14

The catalytic domain has a bi-lobed structure, with an N-lobe and C-lobe connected by a hinge region.
Residues in the N-lobe form contacts with ATP and 2x Mg2+ ions during catalysis. These include:
- Glycine-rich G loop, which connects beta sheets and engages ATP binding
- hinge region
- αC helix, which is able to change its conformation upon activation
The C lobe forms most of the contacts for peptide recognition for phosphorylation → involved in specificity.
The C-lobe catalytic loop, which includes key residues such as the His-Arg-Asp (HRD) motif, coordinates the terminal ATP-𝜸-phosphate in place for transfer to the peptide substrate.
- Acidic residues in the kinase C lobe coordinate the basic and +ve Arg (R) residues in the target peptide at the P-2, P-3 and P-6 positions. These are the consensus sequences that the kinase checks to determine its specificity. The binding pockets hold the arginines in place to allow the phosphorylation reaction to occur → interactions would not occur if arginine was not present at these sites.
The activation loop is the key trigger for kinase activation as it contains the site for autophosphorylation, which drives conformational changes.
The Asp-Phe-Gly (DFG) motif is part of the activation loop. It provides a conformational signature in its orientation of the phenol ring.
- DFGin is associated with the active kinase conformation
- DFGout is associated with an inactive kinase conformation
The catalytic domain has a regulatory (R) spine and a catalytic (C) spine. These are formed from hydrophobic residues in the active kinase conformation.
- The C-spine is completed by adenine base of ATP
- The R-spine contains the Phe in the DFG-in conformation.
- The spines are supported by the hydrophobic αF helix of the C lobe, which provides a base for these.
Autophosphorylation of the activation loop leads to its rearrangement, assembly of the αC helix and binding of ATP, and organisation of the C and R spines ready for phospho-transfer to the substrate.

Question 15

The inactive/active conformation changes are highly dynamic.

Answer 15

Side chains interact with ATP, with the N-lobe and glycine-rich loop forming interactions with adenosine and the phosphates interacting with magnesium ions.
The αC helix performs network interactions to support the ATP binding site.
Phosphorylation of the activation loop drives the process by changing the conformation and causing a global change in the kinase.

Question 16

Regulation of activation

Answer 16

Regulation of catalytic domain activation is highly specific to the individual kinase, with a range of mechanisms, including:
- Phospho-switches: phosphorylation sites in regulatory regions of the kinase
- Binding domains for protein-protein interactions, such as SH2 in SRC, or second messengers, such as Ca2+-calmodulin and the cAMP binding site in the PKA R subunit.
- Dimerization and transactivation - most kinases cannot work on their own

Question 17

Kinase dimerisation

Answer 17

Constitutive dimerisation and activation is seen in kinases like BCR-Abl, where the BCR protein sticks monomers together, causing constitutive autophosphorylation and therefore activation of Abl.
Dimerisation is a key 1st step in autophosphorylation of the activation loop before full kinase activity can occur.
Different proposed dimer modalities exist, including in parallel, via activation loop exchange, or in antiparallel.

Question 18

Intrinsically disordered regions (IDRs)

Answer 18

Intrinsically disordered regions (IDRs) contain short linear motifs (SliMs).
They frequently flank kinase domain cores and play a crucial role in the assembly of heterodimers and integrating kinases into larger regulatory networks.
- Many SLiMs operate as phospho-switches
- IDRs include the activation loop
- Docking motifs in IDRs facilitate dimer assembly

Question 19

Types of small molecular inhibitors for cancer

Answer 19

Type I inhibitors are fully competitive with ATP at its binding site. These drugs tend to be broad-spectrum and non-selective as the ATP site is highly conserved across different families. This creates a risk of side effects and toxicities.
Type II inhibitors also overlap with the ATP site in their binding, but these inhibitors bind an inactive non-phosphorylated form of the kinase in the DFG-out conformation, which exposes an additional allosteric hydrophobic pocket for inhibitor binding. This improves selectivity.
In type III inhibitors, the binding site is adjacent to the ATP binding site.
In type IV inhibitors, the binding site is distant from the ATP binding site.
This can include parts of the catalytic or even regulatory domains.
These types of inhibitors potentially offer:
- Increased selectivity - targeting kinase unique binding sites not conserved in other family members (e.g. regulatory domains)
- Noncompetitive or uncompetitive mechanisms - the high intracellular [ATP] cannot compete against the inhibitor, leading to greater in cell inhibition
- Noncompetitive mechanism - inhibitor does not distinguish between unbound or ATP-bound kinase → reduced Vmax, same Km
- Uncompetitive mechanism - inhibitor binding is enhanced by the additional binding of ATP to the kinase catalytic domain → reduced Vmax AND Km. Inhibitor binding supports the binding of ATP, and vice versa, which seems paradoxical.

Question 20

Examples of different types of small molecular inhibitors

Answer 20

Type I - staurosporine (>180 kinases) - no therapeutic use as it lacks specificity
Imatinib is a type II BCR-Abl inhibitor used to treat CML, but it still inhibits PDGFR and KIT RTKs alongside its target.
Bosutinib has improves selectivity for BCR-Abl.
However, the pharmacology of imatinib has also proven useful - it’s licensed for the treatment of GI tumours where overactivity of both PDGFR and KIT contributes to cancer progression.
Crizotinib is a type II inhibitor designed as a MET inhibitor, a RTK overexpressed in NSCLC. However, during development anti-cancer efficacy appeared more related to inhibition of the EML4-Alk fusion kinase, which is constitutively active. This spurred the generation of more potent Alk inhibitors, such as alectinib, brigatinib and lorlatinib, with better progression-free survival.
Highly selective kinase inhibitors can actually run the risk that the cancer circumvents the inhibition and uses a different mechanism for tumorigenesis, so sometimes multi-target drugs are better, but not quite as non-selective as staurosporine.
Activity against multiple kinases involved in cancer pathways can be beneficial, balanced against risks of side effects or toxicity.

Question 21

Binimetinib - Type III MEK inhibitor

Answer 21

Binimetinib is a type III MEK inhibitor.
Binds the ATP-adjacent pocket formed by the outward movement of the αC helix in the inactive kinase conformation.
Uncompetitive mechanism - ATP can bind concurrently, but it cannot transfer its phosphate. Because of amino acid orientation, Binimetinib binding is actually supported by the binding of ATP, and vice versa.
MEK is involved in the MAPK cascade. Targeting MEK selectively may inhibit effects of multiple upstream pathways in cancer.

Question 22

Asiminib - Type IV Abl inhibitor

Answer 22

Native Abl 1a/1b has a role in cell cytoskeletal remodelling, DNA repair and apoptosis.
Physiologically expressed Abl 1b demonstrates N-terminal myristoylation, a fatty acid modification.
Myristate (myristic acid) negatively regulates the kinase catalytic domain by binding a pocket in the C lobe and switching off the kinase in the absence of appropriate activation stimuli.
In BCR-Abl, the myristoylate modification, but not its C lobe binding site, is lost. This constitutively switches on the enzyme, driving dimerisation and autophosphorylation.
Asiminib is a type IV BCR-Abl inhibitor that binds at the myristate binding pocket between the αE, αF, αH helixes - this makes it highly selective.
Binding unlocks the inactive conformation of the kinase.
It has preserved activity against BCR-Abl resistant mutants.

Question 23

Targeting Kinase mutation in cancer: B-Raf

Answer 23

The B-Raf V600E mutation is found within the activation loop of 50% of patients with melanoma. It is also present in other cancers, like NSCLC.
This valine to glutamate mutation substitutes for the autophosphorylated Thr599/Ser602 trigger as glutamate brings in the same negative charge caused by phosphorylation, constitutively activating the kinase.
There is consitutive B-Raf activity and activation of the pro-proliferative MAPK pathway.
This led to the design of ATP-binding site inhibitors with higher affinity for the V600E stabilised (active) conformation, such as vemurafenib and dabrafenib, so-called type 1.5 inhibitors.
Vemurafenib was the first drug approved to treat B-Raf mutant metastatic melanoma. It can stabilize the inactive conformation of B-Raf, preventing dimerization and activation.
It has 10-fold in vitro biochemical affinity for the mutated B-Raf. However, looking at phenotypic assays, we can see that selectivity is much HIGHER in vivo, resulting in a reduction in tumour size due to oncogenous addiction.
Vemurafenib has a dramatic therapeutic effect in late–stage melanoma.
There are however some limitations, including:
- Other classes of B-Raf mutations are not sensitive to these inhibitors
- Paradoxical potentiation of WT B-Raf dimerisation/activation in other cells can occur, especially when Ras-MAPK is already activated by RTKs, leading to skin rash and squamous cell carcinoma.
- Resistance often develops within 12 months due to upregulation of alternative signalling pathways and the acquisition of new mutations, but there is still an increase in progression-free survival.

Question 24

Kinase inhibitor group selectivity

Answer 24

Some compounds exhibit “group selectivity”, meaning they broadly interact with kinases within a specific subfamily while remaining selective outside that group.
Testing compounds against kinases closely related to the intended target does not reliably indicate overall selectivity, as group-selective inhibitors can broadly interact with members of the targeted kinase group but not with other groups. This challenges the traditional approach to assessing selectivity.

Question 25

Rational design of type II inhibitors **

Answer 25

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.

Question 26

Inhibitor resistance in cancer

Answer 26

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.

Question 27

Resistance mutations at the target

Answer 27

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.

Question 28

Covalent inhibition (Type V)

Answer 28

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.

Question 29

Analysis of covalent inhibitors

Answer 29

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.

Question 30

Framework for covalent inhibition

Answer 30

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&raquo_space;> 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.

Question 31

Jump dilution

Answer 31

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?

Question 32

PROTACs

Answer 32

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.

Question 33

Rational design of covalent inhibitors

Answer 33

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.