10A-10C Kinases - Lecture notes 10 PDF

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CHE306U ADVANCED PHARMACEUTICAL CHEMISTRYKINASE INHIBITORSLearning Objectives- Describe the role of a protein kinase and how ATP binds to the kinase - Discuss how inhibitors can bind to the ATP binding site and achieve selectivity - Describe a Type 1 kinase inhibitor - how EGFR inhibitors were optim...

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CHE306U ADVANCED PHARMACEUTICAL CHEMISTRY KINASE INHIBITORS Learning Objectives -

Describe the role of a protein kinase and how ATP binds to the kinase Discuss how inhibitors can bind to the ATP binding site and achieve selectivity Describe a Type 1 kinase inhibitor - how EGFR inhibitors were optimised and their clinical uses Describe a Type 2 kinase inhibitor - Evaluate and discuss the discovery and binding of imatinib to Bcr-Abl including the mechanism of resistance Describe additional kinase strategies; covalent inhibitors and multi-kinase activity Design analogues of kinase inhibitors based on knowledge of SAR

VIDEO 10A: KINASE INHIBITORS PART 1 The phylogenetic tree of kinases (The kinome)

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The human kinome is made up of lipid kinases and protein kinases; the vast majority of kinases are in the section of protein kinases Around 538 protein kinases are encoded in the genome and some common examples are the tyrosine kinases and the tyrosine like kinases at the top of the tree and they have been frequently targeted by various drugs

The role of protein kinases -

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Protein kinases catalyse the covalent addition of phosphate groups to target proteins and this event represents a central mechanism for regulating cellular and enzymatic function Role of kinases: transfer a phosphate group to a hydroxy group on a protein – this is mediated by a kinase and that converts ATP into ADP and the phosphate is transferred to the hydroxy group on the protein and then that protein becomes

phosphorylated o The reversed process is catalysed through phosphatases where the phosphate group is removed

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This process is extremely important in nature where kinases are key controllers of most biochemical pathways and therefore are important in regulating health and disease There are actually more than 160 protein kinases associated with disease in humans, so they make good drug targets and actually again greater than 20 kinase inhibitors are drug targets that have been looked at in clinical development or have realised a marketed drug o They are a really important target class for treating diseases

Protein Kinases – the role of ATP

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If we look at the role of ATP and protein kinases, as we have seen kinases transfer a phosphate to specific amino acids in the protein and those residues will contain hydroxy groups such as tyrosine serine or threonine, but also histidine which are present in bacteria o We can see the structures above; ATP gets converted to ADP and that phosphate group that is released is transferred onto a particular amino acid with the hydroxy residue Enzymes that catalyse phosphorylation reactions on protein substrates Tyrosine kinases, serine-threonine kinases (amino acids with OH group to be phosphorylated) ATP used as the enzyme cofactor - phosphorylating agent

Why is phosphorylation important?

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Phosphorylation is very important; on the left-hand side, some of the processes where kinases regulate the activity in cells have been listed, e.g., proliferation of cells or apoptosis of cells are very important, but also processes such as differentiation of cells or division of cells can be added to the list

Protein kinases and kinase linked receptors -

Protein kinase receptors - dual role as receptor and enzyme o Bind an external messenger (growth factors and other locally released proteins that are present at low concentrations) o Intracellular kinase active site

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Protein kinases are enzymes which exist in the cytoplasm but there are also protein kinases which transverse the cell membrane and can act as both a receptor and an enzyme To highlight, look at picture 1 on the top left: the kinase is in the closed conformation and in a resting state, but upon binding of a particular messenger such as a hormone for example, this binds to the receptor on the outside of the cell that changes the protein conformation and the enzyme is now in the active conformation and that then allows phosphorylation to occur, so that takes place in picture 3, where the messenger is bound and converts the kinase from the closed form into the active form and then that allows phosphorylation to occur

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Protein Kinases Protein kinases are also present in the cytoplasm and these tend to be regulatory proteins which play key roles in cell proliferation and survival, for example and the difference here for these proteins is that they are without a transmembrane receptor and are the subjects importantly for anti-cancer drug targets and we can see on the left a plot of the number of new chemical entities approved, which are kinase inhibitors, and the years that they were approved, and you can see there in the light blue that most of the disease targets are in the cancer field which is oncology and very few outside of that field, so kinases again are key targets in the cancer field for treating various diseases in that area o Cytoplasmic Tyrosine Kinases vs. Receptor Tyrosine Kinases o Signal transduction processes Signalling cascades control gene transcription leading to cell growth and division Overexpression can result in cancer -

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Tyrosine Kinases -

Phosphorylation reactions on tyrosine

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If we look at protein kinases and what they do with the particular residues, they can be divided into 2 groups o First, we have tyrosine kinases as shown here where a phosphate is added to the hydroxy group in the tyrosine to give the phosphate o Secondly, phosphorylation reactions can occur on serine or threonine

Serine/Threonine Kinases -

Phosphorylation reactions on serine or threonine Secondly, phosphorylation reactions can occur on serine or threonine and these are carried out by serine or threonine kinases, so again, quite simply a phosphate group is attached to the hydroxy group of those particular residues

Kinase Active Site -

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Contains the binding site for the protein substrate o Has to accommodate the protein substrate to allow phosphorylation to occur Contains the binding site for the ATP cofactor o Actually, all kinases contain an ATP binding site close by Clinically useful inhibitors target the ATP binding site o If that is inhibited, that can have some really profound effects on treating disease ATP binding site is similar but not identical for all protein kinases o One of the problems with kinase inhibitors is the similarity in the ATP binding site between all of the kinases previously mentioned; there are over 500 kinases all with an ATP binding site, so how to you gain selectivity for each of the different kinases? So that is actually one of the issues o But it IS possible to induce selectivity for various kinases in particular ways Allows selectivity of inhibitor action o It IS possible to induce selectivity for various kinases in particular ways Much of the understanding into kinases comes from crystallographic studies, X-ray docking pictures where you can see the inhibitor bound to the target and you can see all the interactions and actually interactions where you can improve the inhibitor design

ATP binding site If we focus in on the ATP binding site, there are various aspects to that and in this case, what we have is ATP (natural ligand) bound into the kinase ATP binding site and what we can see firstly on the top left is the region called the hinge region which is a very important region because that forms key hydrogen bonds with ATP here (2 have been drawn, but there are probably more) - Secondly, we have the adenine heterocycle in ATP there, which makes Van der Waal force interactions with the protein and that increases the affinity of the ligand into the kinase Also, we have a ribose pocket which binds the sugar of ATP, and we also have a cleft region to the right of that which can take the polar phosphate group and actually that binds magnesium ions and that area is also moved towards the surface and there is a bit more solvent exposed If we look at the top right of the diagram, we have a hydrophobic pocket as opposed to a hydrophilic pocket in the cleft and that hydrophobic pocket is very useful for gaining some selectivity in particular inhibitors and then just adjacent to that it is important that we have the gate keeper residues which guard the entrance to the pocket and its size is important in drug design -

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ATP binding site To look at a holistic model on the right-hand side there, you can see the 2 lobes of the kinase; we have the upper lobe on the top and the larger bottom lobe below it and in the middle, we have the hinge region where ATP is bound, and that is typically where drug inhibitors bind as well, displacing or preventing ATP from binding in the same ATP binding pocket - It is also important to mention that there is also a P-loop in the kinase (it is not showing but it is important to recognise that it controls the active and inactive conformations of the kinase and it can move depending on whether it is phosphorylated or not itself, and that can move dependent on that process and what it does do is alter this loop, which is called the DFG loop, so that is a tripeptide sequence (aspartic acid, phenylalanine and glycine) and that loop can move inwards or outwards depending on the activation of the P-loop and it is actually possible to design kinase inhibitors for both the active and the inactive forms, which is quite useful because in the inactive form, the binding site can reveal an additional hydrophobic loop to allow drugs to bind to and that potentially gain extra activity and selectivity for a particular kinase -

Types of kinase inhibitors

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Brief overview of types of kinase inhibitors, so we have the type 1, 2, 3, and 4 Type 1: the ATP competitive site and is the active conformation of the kinase and the DFG sequence is pointing inwards to the ATP binding site; that differs in the Type 2 Type 2: recognise the inactive conformation and we have the tripeptide sequence DFG loop pointing outwards Type 3 and 4: generically termed allosteric because they bind directly where ATP binds, so they can be inhibitors that bind adjacent to the ATP binding site, as in type 3 or quite far away from the binding site as in type 4

Most approved protein kinase inhibitors are Type 1

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If we look at the most approved protein kinase inhibitors that have been marketed, the first compound marketed was imatinib in 2001, which actually was a type 2 kinase inhibitor, but if you look more recently from 2014 onwards, you can see a lot of dark blue there, which are type 1 kinase inhibitors There are also covalent inhibitors coming to the 4

Type I Kinase Inhibitors -

Type I inhibitors act on the active conformation of the enzyme Type I inhibitors bind to the ATP binding site and block access to ATP o Block binding of substrate also

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Can see some similarities to ATP with the adenine structure being replicated here with aminoquinazoline template cores and you can see some other groups as appendages coming off that template which allow these compounds to bind and be competitive with ATP itself

Type I kinase inhibitor -

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Example of a type 1 kinase inhibitor bound to a kinase active site and again we can see the various regions so we have the ATP binding site in the middle where ATP would normally reside; this compound blocks the ATP and that’s why it is an inhibitor We have hydrophobic pocket 1, hydrophobic pocket 2 that the compound can bind to and importantly we have the hinge region which show

these hydrogen bonding networks o There is also the gate keeper at the top, which is important whereby that’s important to control the size of the inhibitor that’s allowed to bind into the site Type II Kinase Inhibitors If we look at the type 2 kinase inhibitors, by first inspection you see the compounds look quite similar, so we have 2 examples here; imatinib and sorafenib, but importantly it is showing that these compounds bind to the kinase to stabilise the inactive conformation so that DFG loop is pointing outwards, but very importantly, in the inactive conformation, there is the opportunity to bind to a hydrophobic pocket in a bit more detail and forms greater interactions compared to type 1 inhibitors and the problem with that is whilst you could generate greater selectivity for type 2 inhibitors, the hydrophobic pocket is quite close to the gate keeper and that is where quite a lot of the mutations occur and that could eventually weaken the binding attractions of any inhibitor that is designed and it could lead to drug resistance Type II inhibitors bind to the enzyme and stabilise the inactive conformation o The hydrophobic pocket is exposed directly adjacent to the ATP binding site Type II inhibitors have the potential to be more selective, but there is a greater risk that random mutation of the target will weaken binding interactions and lead to drug resistance -

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Type II kinase inhibitor -

Here again is a binding network of compound docked into the kinase inhibitor and this time we can see the compound has now really penetrated the hydrophobic pocket 2 and also even moving even further out of the ATP binding site to another allosteric type site and you can see now for this particular compound it is bound into the kinase, hydrophobic pocket 1 is not bound so the

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compound is clearly binding in a different set of sites with different binding interactions compared to a type 1 inhibitor Another point to note for a type 2 kinase inhibitor is that we have now got opportunity to form a network of hydrogen bonds in the allosteric site with polar residues and that can also help improve the physiochemical properties by having water stabilising groups and also we have the amide in red that forms a hydrogen bond network interaction as well and anchors the molecule into the hydrophobic pocket

Kinase inhibitors – summary so far… -

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SAQs

Clinically used inhibitors mimic binding interactions of ATP (adenine part in particular) o 2/3 H-bonds around the hinge region + vwd Developing a selective kinase drug is still a challenging task due to the high similarity of the ATP binding sites across the whole kinome in the ATP binding site but extra sites and hydrophobic pockets are being revealed around the ATP binding site which can allow an increase in selectivity Selectivity can achieved by designing interactions with regions not occupied by ATP o Hydrophobic pockets or gate keeper Only a few protein and lipid kinase targets (about 80) out of the 500+ protein kinases in the human kinome have been successfully targeted so there remains a huge opportunity to target further kinases to combat disease Most of the kinase inhibitor drugs are used for oncological indications Many kinase inhibitor drugs are used to target the same indication (mainly due to the generation of resistance) so if drug A for example develops due to a mutation in the kinase then another compound needs to be designed that overcomes that mutation in the receptor and therefore could be active and then that process keeps going on and on, so there will be multiple generation of kinase inhibitor drugs for the same disease

VIDEO 10B: KINASE INHIBITORS PART 2 Epidermal growth factor receptor (EGF-R) – Type I inhibitors -

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Also known as human epidermal growth factor receptor 1 (HER1): the kinase inhibitor in question will act on this growth factor Membrane bound tyrosine kinase receptor involved in cellular growth o One of 4 HERs (HER1, HER2, HER3, HER4) o Is a tyrosine kinase receptor which means that it has an extracellular binding site for epidermal growth factor and it also has an intracellular kinase site for phosphorylation EGF is a bivalent ligand which binds to 2 receptors at the same time and that causes the receptor dimerization Ligand binding gives formation of homodimer Leads to autophosphorylation of multiple tyrosine residues Adaptor proteins recognise phosphorylated receptor and initiate signalling leading to: o Cell proliferation via Ras/MAPK pathway o Cell survival through antiapoptosis signals (PI3K, AKT)

Epidermal growth factor receptor (EGF- R) -

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Overexpression of EGFR associated with breast, lung, brain, prostate, GI and ovarian cancers o This body of evidence implicating over-expression of the receptor as a tumour driver has prompted a variety of approaches to target tumours using EGFR inhibitors o These high expression levels have been correlated with poor prognosis and more rapid disease progression, certainly for cancer and so this evidence is linked with overexpression to the growth of tumours and so the approach here to target tumours is to block the EGFR receptor through kinase inhibition and actually several drugs have been marketed as EGFR inhibitors, so for example Gefitinib and Erlotinib have been approved for various cancers Gefitinib was approved for treatment of refractory lung cancers Erlotinib approved for non-small cell lung cancer Drug resistant tumours so clearly the drugs are having reduced effect even though they bind and that typically occurs in the gatekeeper region where mutations are likely and that controls entry of the molecule to the ATP binding site and a mutation there can sometimes reduce the level of binding of the inhibitor o Gatekeeper mutation o Competitive inhibitor still able to bind but less effective

Epidermal growth factor receptor (EGF- R) If you look at the mechanism of action of how epidermal growth factor works, we can see here it is in the cell membrane – it is a transmembrane and it exists there as the inactive EGFR monomeric receptors and on the extracellular side you have epidermal growth factor, which is a bivalent ligand, which binds 2 of the EGFR receptors, and when it does it causes ligand dimerisation and that then causes a conformational change in the dimerised receptors and that then opens the kinase to the active form and then the free hydroxyls and tyrosine in this case are able to be phosphorylated by the tyrosine kinase and you can see the phosphorylated kinase on the right hand side and it is this phosphorylation process which is thought to be believed to cause tumour proliferation so any inhibition of this process by a kinase inhibitor should be able to reduce the prevalence of tumour -

Identification of Type I inhibitors of EGF- R Rate studies showed that EGFR forms a ternary complex with ATP and the peptidic substrate - Define a query used to interrogate a database of predicted 3D chemical structures - If you look at how to identify this type 1 inhibitors of this particular kinase, Astra Zeneca, a large pharmaceutical company conducted a virtual screening approach, looking at likely interactions of a ternary complex with EGFR, ATP and the peptic substrate that was going to be phosphorylated and they did some computer modelling and docking and came up with a pharmacophore and they interrogated their own database; they cherry picked a subset of the screening collection, then screened those compounds and they identified several hits with inhibitory activity in a cellular assay and after a small amount of SAR was generated through chemistry, they converted the top compound, which is the chlorophenyl compound into the methyl group and also introduced the dimethoxy groups and that revealed the first potent lead for the kinase inhibitor o Searching for motifs that mimic that ATP γ-phosphate and the tyrosine phenol, key features of this complex catalytic system o 6 membered aromatic ring to mimic ring of tyrosine o Oxygen or nitrogen at a suitable distance to mimic phenolic OH o Oxygen or nitrogen at a suitable distance to mimic non-bridging oxyge...