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Structure Defines Function – Clinically Relevant Mutations in ErbB Kinases Janina Niggenaber, Julia Hardick, Jonas Lategahn, and Daniel Rauh J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00964 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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Journal of Medicinal Chemistry

Structure Defines Function – Clinically Relevant Mutations in ErbB Kinases Janina Niggenaber,‡ Julia Hardick,‡ Jonas Lategahn,†,* and Daniel Rauh* Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund (Germany). Drug Discovery Hub Dortmund (DDHD) am Zentrum für Integrierte Wirkstoffforschung (ZIW), 44227 Dortmund (Germany). KEYWORDS: Cancer, Targeted Therapy, Tyrosine Kinase, Drug Resistance, Structural Biology

ABSTRACT: The ErbB receptor tyrosine kinase family members EGFR (epidermal growth factor receptor) and Her2 are among the prominent mutated oncogenic drivers of non-small cell lung cancer (NSCLC). Their importance in proliferation, apoptosis, and cell death ultimately renders them hot targets in cancer therapy. Small-molecule tyrosine kinase inhibitors seem well suited to be tailor-made therapeutics for EGFR-mutant NSCLC; however, drug resistance mutations limit their success. Against this background, the elucidation and visualization of the three-dimensional structure of cancer-related kinases provides valuable insights into their molecular functions. This field has undergone a revolution since X-ray crystal structure determinations aided structure-based drug design approaches and clarified the effect of activating and resistance-conferring mutations.

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Here, we present an overview of important mutations affecting EGFR and Her2 and highlight their influence on the kinase domain conformations and active site accessibility.

Introduction Lung cancer has the highest cancer incidence and mortality worldwide.1 The identification of predictive biomarkers and a detailed understanding of drug resistance recently led to a substantial boost in the development of selective small-molecule inhibitors to specifically inhibit oncogenic targets. To selected patient populations, this precision medicine offers an alternative to platinumbased chemotherapy with improved survival and enhanced quality of life.2, 3 Genetic mutations affecting the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and Her2, members of the ErbB receptor tyrosine kinase family, result in the onset and progression of non-small cell lung cancer (NSCLC) or drug resistance to targeted small-molecule inhibitors. These targets comprise an extracellular ligand-binding domain, a transmembrane segment, and an intracellular tyrosine kinase domain.4, 5 Upon ligand binding, EGFR undergoes homo- or heterodimerization with Her2, for example.6 Autophosphorylation of tyrosine residues within the intracellular kinase domain leads to complete activation.5, 7 Activated EGFR is part of important signal transduction pathways, such as RAS–RAF–MEK–ERK or PI3K–AKT–mTOR, regulating proliferation, apoptosis, and cell death, among others. Dysregulation of EGFR or Her2 signaling therefore ultimately leads to tumorigenesis.4, 5, 8 For the treatment of aberrant ErbB signaling, three generations of targeted inhibitors are readily available and currently in clinical use (Figure 1C and Figure S1). First-generation tyrosine kinase inhibitors (TKIs) comprise ATP-competitive inhibitors such as gefitinib9, Afatinib12,

13

and dacomitinib14,

15

10

and erlotinib.11

are second-generation inhibitors with an acrylamide that


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covalently reacts with a reactive cysteine in EGFR and Her2. In this way, competition with the cofactor for the binding site is offset, the drug–target residence time is maximized, and selectivity within the kinome is increased.16 Although similar to first-generation inhibitors, this warhead is the only distinctive feature. Therefore, adverse events related to wild-type inhibition which are observed with first-generation TKIs are similarly observed with second-generation EGFR inhibitors. Lapatinib17 and neratinib18-20 are first- and second-generation inhibitors, respectively, that bind to inactive EGFR, as outlined below. Inhibitors in the third generation are best exemplified by osimertinib,21-23 rociletinib,24 and olmutinib,25 which also incorporate an acrylamide but are based on different scaffolds that allow for a high degree of mutant selectivity. To be precise, these inhibitors are specifically designed to target mutant variants of EGFR, while sparing wild-type.26 In the quest to optimize patient selection or develop a fourth generation of inhibitors targeting (multi-)mutated EGFR, a detailed understanding of how mutations influence its activity and drug susceptibility is required.8 Against this background, structure determination methodologies are key to gaining detailed insights into the receptor’s molecular features at the atomic level. Among these methods, protein X-ray crystallography has yielded several datasets that allow for dissecting the effect of distinct mutations on the kinase domain of EGFR and Her2. Here, we review from a structural perspective the relevant activating and drug-resistant mutations affecting the ErbB kinases and their influence on conformation and active site texture.


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Discussion EGFR Kinase Domain Conformations Predict the Activation State The kinase domain of EGFR is subject to an equilibrium between inactive and active conformations (Figure 1A). It is tightly regulated by extracellular ligand binding,27,

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dimerization,29 internalization,30, 31 and degradation.32, 33 Upon a ligand-binding event, receptor dimerization is initiated, resulting in asymmetric dimer formation of the intracellular kinase units. This formation allows interaction of the C-terminus of the activator kinase with the N-terminal portion of the receiver kinase. The inactive receiver kinase is characterized by an outwards rotated helix C (Figure 1A, red), which allows the activation loop (orange) to form a short helical segment next to the helix C.27, 34 This kinase conformation facilitates binding of inhibitors, such as lapatinib or neratinib,35 which can be assessed by a direct binding assay utilizing a fluorescent labeled activation loop.36 The interaction with the activator kinase forces a rearrangement of the receiver kinase, resulting in its active state. The activated conformation exhibits an extended activation loop (blue), stabilized by a short antiparallel -segment, to allow inward rotation of helix C (green), which creates the interaction surface with the activator kinase. In addition, Glu762 forms a characteristic salt bridge with the catalytic Lys745 side chain. This partially active dimer becomes fully activated following trans-autophosphorylation, as mentioned above.27, 34, 37 Of note, crystallographic studies have revealed different conformational states with respect to the DFG triad (DFG-in active,38 Src-like (DFG-in/helix C-out) inactive,39 and DFG-out inactive40).

Point Mutations Within the Activation Loop Destabilize the Inactive Helix C-Out State Given the importance of the regulatory helix C, it is conceivable that mutations affecting its conformational freedom occur in cancer. Because of their shift in kinase conformation equilibrium


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in favor of the active state, these aberrations are termed “activating mutations”.41 One of the most prevalent EGFR activating mutations found in NSCLC patients is the substitution L858R in exon 21, occurring in around 38% of cases.42 It is located within the activation loop, central to the crucial helical turn structure (Figure 1B, orange). A polar interaction between the main chain carbonyl of Leu858 and the main chain nitrogen of Leu862 contributes to stabilization of the inactive helical turn motif. Through packing into a hydrophobic cavity between the helix C and the glycine-rich loop (shown in red and gray, respectively), the Leu858 side chain takes a pivotal role in stabilizing the inactive state. Specifically, the residues Leu858, Leu861, and Leu862 (activation loop), Leu788, Leu777, Met766, Ile759, and Leu747 (helix C and adjacent regions), and Phe723 (G-rich loop) are involved. Within this arrangement, the mutated, much larger, and charged Arg858 side chain would disrupt the hydrophobic assembly and is incompatible with the inactive state. Therefore, it shifts the kinase domain towards the active conformation by resolving the important helical turn structure and providing space for the helix C to rotate inwards. In addition, Arg858 further stabilizes the active state of the mutant kinase, forming a network of polar interactions with Arg836 and Tyr891 (Figure 1B, blue).38 The mutation L861Q was also observed in cancer patients,43 and as being located within the helical turn motif next to Leu858 and participating in the hydrophobic cluster (Figure 1B, orange), is proposed to activate EGFR via a similar mechanism.38 Together, the point mutations L858R and L861Q trigger a shift in the equilibrium to a constitutive active kinase conformation, which can be clearly assessed through crystal structures obtained from wild-type and L858R-mutant kinases. These findings are directly relevant because affected cancer patients respond well to treatment with approved small-molecule inhibitors.44, 45


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Figure 1. Inactive and active kinase domain conformations. (A) The inactive conformation (bound with lapatinib; PDB ID: 1XKK) exhibits an outwardly rotated helix C (red) and a small helical turn within the activation loop (orange). It is in an equilibrium with the active conformation (bound with erlotinib; PDB ID: 1M17), which exhibits an inwardly rotated helix C (green) that allows for a salt bridge between the catalytic lysine Lys745 and Glu762. Moreover, it is characterized by an extended activation loop (blue). (B) The side chain of Leu858 within the helical turn (orange)


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plays a crucial role in stabilizing the inactive conformation by packing into a hydrophobic cavity between helix αC (red) and glycine-rich loop (gray). Mutation of this amino acid to the polar Arg858 (PDB ID: 2ITT) therefore results in an equilibrium shift toward the active conformation. The QR-codes provide an augmented reality view of a corresponding 3D model.46 (C) Chemical structures of representative examples of the three TKI-generations of EGFR.

Deletion Mutations in Exon 19 Restrict the Flexibility of the Regulatory Helix C Another subgroup of TKI-responsive tumors exhibits exon 19 deletion mutations affecting the β3–αC protein strand adjacent to the helix αC. These mutations are observed in 46% of patients and are the most prevalent and clinically important mutations beside the point mutation L858R. The deletion mutations, as well as the activating point mutation, cause the constitutive activation of the kinase by destabilizing the inactive conformation, which is maintained absent of ligand stimulation.42, 44, 45, 47 Foster et al. analyzed public available data sets and reported the clinically most frequent deletion mutation of the ELREA motif (delE746-A750), followed by a lower prevalence of delREAT and delLRE.48 Homologous short in-frame deletions within the Her2 kinase domain (delLRENT) are reported in Her2 non-amplified breast cancer at a low frequency.49 In this context, attempts to gain crystal structures of respective mutants have been unsuccessful to date. Therefore, insights were attained by means of in silico molecular dynamics (MD) simulations, which add up to the repertoire of structure elucidation methodologies. The ELREA motif plays an important role in the transition between the active and inactive conformations of the regulatory helix αC and promotes the required flexibility of the region.50 Modeling studies suggest that the deletion induces partial


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loss of helicity within the N-terminal portion of helix C, which is repositioned to form a tight β3– αC loop while retaining the characteristic Glu762–Lys745 salt bridge (Figure 2). It is therefore inferred that the reduced length and flexibility of this strand locks the helix αC-in state and prohibits transition to the inactive conformation to promote an active kinase domain.48, 51 This assumption is underlined by the different sensitivities of the EGFR mutants to the inhibitors erlotinib and lapatinib. Erlotinib binds to the active helix αC-in conformation. Accordingly, the mutants show a significant sensitivity relative to wild-type EGFR. In contrast, lapatinib binds to the helix C-out conformation, and resistance is associated with deletion mutants of EGFR.48 Similarly, the recently reported allosteric EGFR TKIs failed to target deletion mutations in EGFR.52 In agreement, further studies have emphasized that reducing the length of the β3–αC loop shifts most low-energy conformations accessible to the αC helix from the out-conformation towards the in-conformation. The deletion of five amino acids leads to the optimal helix αC orientation for catalytic activity, while deletion of six or more amino acids leads to structural perturbations that diminish kinase activity. Of interest, the deletion of six residues can trigger an active kinase if a serine replaces a rigid proline side chain N-terminal at the beginning of the helix αC (P753S; Figure 2) to allow for a more extended conformation. Conversely, shorter deletionlength mutations promote an active kinase domain if accompanied by substitution of a remaining loop residue to a proline.48 Taken together, these data illustrate that EGFR β3–αC deletions in exon 19 shift the equilibrium to the active helix αC conformation, which remains inhibitor-sensitive. However, it is incompatible with TKIs which bind to helix C-out kinase conformations.


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Figure 2. Deletion mutation of the ELREA motif in exon 19. MD simulations (blue) suggest that these mutations lock the kinase in its active conformation (green; PDB ID: 1M17) by restricting the flexibility of helix αC, which is crucial for adopting the inactive conformation (black outlined; PDB ID: 2JIV).

Insertion Mutations in Exon 20 Heterogeneously Respond to TKIs A small subset of patients (9%) diagnosed with lung adenocarcinoma presented with in-frame insertion mutations within exon 20 of EGFR.42 Contrasting with exon 19 deletions and L858Rbearing tumors, most mutations of this type are considered primary resistant because current treatment options are limited to chemotherapy and clinically effective small-molecule TKIs are lacking.53 Clinically relevant insertion mutations primarily occur adjacent to the helix αC within the αC–β4 loop. The mutations D770_N771insSVD and V769_D770insASV together account for 36% of EGFR exon 20 insertions, resulting in duplication of codon 768–770 or 767–769, respectively (Figure 3A).42, 54 The understanding of the molecular features of this type of mutation is limited; moreover, crystallographic studies have yielded only one crystal structure so far. The only co-crystal structure reported revealed EGFR-D770_N771insNPG bound with the covalent


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quinazoline-based second-generation inhibitor PD16839355 at a poor resolution of 3.5 Å.53 The insertion mutation lies at the C-terminal end of the helix αC immediately following Asp770, which forms a tight turn together with the three inserted residues. In this assembly, a hydrogen bond formed between the main chain carbonyl of Asp770 and the amide of the inserted glycine locks the kinase in its active conformation by keeping the helix αC in its inward rotated position (Figure 3B).53 After the insertion, the protein chain is quite similar to active EGFR kinase domains.53, 54 Based on inspection of this crystal structure, Kosaka et al. proposed that the residue Asp770 plays a key role because of its localization in a region that is rearranged in the translocation between the active and inactive conformations. During transition to the inactive conformation, the side chain of Arg776 is repositioned to form hydrogen bonds with the backbone carbonyl groups of Ala767 at the end of the helix αC and Leu703. Although this position is available in wild-type EGFR, the inserted residues reposition Asp770, sterically blocking Arg776 from accessing the end of the helix αC (Figure 3B). In accordance with this idea, exon 20 insertions that replace Arg770 with a flexible glycine (D770delinsGY; Figure 3A) are sensitive to afatinib because of restored access of Arg776.54 The inserted residues are not directly in contact with the ATP binding site, however, it has been speculated that drug resistance may arise from a narrow binding site that is incompatible with third-generation TKI binding.56 Yasuda et al. analyzed a homology model of A763_Y764insFQEA-mutated EGFR. Contrasting with previously described exon 20 mutations, which affect the αC–β4 loop or the beginning of helix αC, these four amino acids are inserted central to the regulatory helix C (Figure 3A). Moreover, patients harboring this insertion mutation remain sensitive to TKI treatment. The FQEA sequence is expected to form one additional helical turn and shift the register of the adjacent residues towards the N-terminus. In this way, the catalytically important Glu762 is displaced by


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one turn and effectively replaced with the glutamic acid residue introduced by the insertion. More important, the mutation places a smaller alanine side chain in the former position of Ile759. Again, the amino acid at this position is involved in a hydrophobic interplay around Leu858 (Figure 1B), which is indispensable to stabilizing the inactive state. Here, a weakened interaction accounts for sensitivity towards small-molecule inhibitors. Moreover, it can be speculated that a I759A mutation might occur, eliciting a similar effect.53 Hirano et al. conducted further studies of the mutation Y764_V765insHH (Figure 3A), using homology modeling. They assumed that the catalytically essential Glu762 retains its position while shifting the inserted residues toward the C-terminus. In this way, two histidine residues are inserted in a distinct position, one directly interfering with the binding of anilinoquinazoline-based inhibitors and the second interrupting the hydrophobic cavity with Leu858. As outlined above, Leu858 takes a prominent role in stabilizing the inactive state and is prone to mutations, preserving its active conformation. However, third-generation TKIs remain resistant to this type of exon 20 insertion mutation. This finding is not explained by the model presented, highlighting the need for further structural characterizations.57 Of note, mutations in Her2 show structural similarity to EGFR exon 20 insertion mutations. However,

the

observed

Her2

insertion

mutations

are

less

homogeneous,

with

A775_G776insYVMA (resulting in duplication of codons 772–775) accounting for 80% of mutations found in Her2-mutant NSCLC (Figure 3C).6 Data suggesting success of treating Her2mutant tumors with TKIs are limited to date.54 Crystallographic studies of Her2 focus on the extracellular domain and their targeting with antibodies or related approaches. Two crystal structures of the wild-type Her2 kinase domain are deposited in the protein data bank (PDB) and show a flexible GVG motif adjacent to the regulatory helix.58, 59 This structure contrasts with other


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ErbB kinases, which exhibit an SVD sequence in the homologous position (Figure 3A and 3C). Her2 insertion mutations are located next to this flexible motif, leading Aertgeerts et al. to propose their role in stabilizing the loop region, resulting in reduced flexibility of the helix αC.58 Taken together, considerations on the structural level imply an effectiveness in treating a subset of tumors in the clinical setting with appropriate EGFR-targeted TKIs. Although most exon 20 insertion mutations remain resistant and their treatment challenging, D770delinsGY and A763_Y764insFQEA are sensitive to approved inhibitors as predicted by 3D models. The latter one exhibits sensitivity because it interferes with the hydrophobic assembly, similar to the L858R mutation. Further structural characterizations will certainly unravel the features of elusive exon 20 mutants affecting codon 767–775 in EGFR and Her2.


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Figure 3. Insertion mutations in exon 20. (A) Overview of the clinically most prevalent EGFR insertion mutations in exon 20 (PDB ID: 1M17). (B) A crystal structure of EGFRD770_N771insNPG (blue; PDB ID: 4LRM) revealed a small wedge that locks the kinase in its active conformation by hindering Arg776 to stabilize the inactive conformation (red; PDB ID: 1XKK). (C) Overview of the clinically most prevalent Her2 insertion mutations in exon 20 (PDB ID: 3PP0).

Point Mutations Within the Glycine-Rich Loop Influence Its Crucial Dynamics Among the structural elements comprising the kinase domain, the glycine-rich loop (homologous to the P-loop in phosphatases) plays a crucial role during ATP or ligand binding. This highly conserved and flexible element forms like a lid over the active site and is characterized by a canonical GxGxxG sequence pattern, in which x represents any amino acid. In EGFR, this sequence is located in exon 18 as GSGAFG (Figure 4). Because of its vital influence on the active site, it has a major effect on the affinity of TKIs. Thus, it is not surprising that point mutations in this region affect ligand-binding events.60, 61 Accordingly, mutations in Gly719, which is the first glycine within the G-rich loop, are found in about 2% of NSCLC patients.42 Mutations replacing the glycine at codon 719 by Ser, Cys, or Ala side chains are most frequently detected,38,

62-64

and exhibit sensitivity towards first- and

second-generation TKI treatment.44, 45 Although the mutation G719S was characterized by means of X-ray structure determinations and yielded several PDB entries (Figure 4), its effect is not yet understood in detail. In line with reports by Yun et al., Yoshikawa et al. revealed a slight upwards shift of the Gly-rich loop, which was proposed to account for observed reduced affinity towards ATP and gefitinib.38, 65 Because the cofactor and ligand compete for the binding site and their loss


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in affinity is not equally high, gefitinib binds 6-fold more effectively to the binding site, as compared to wild-type EGFR. In addition, a stabilizing effect of G719S on the active kinase domain conformation has been assumed because the adjacent Phe723 is part of important interaction networks contributing to a stabilized inactive conformation, as mentioned above.38 Accordingly, the influence of this central phenylalanine was assessed by analysis of the F723A mutation, which showed a higher gefitinib-sensitivity compared to wild-type or the G719S mutation.65 These considerations are in line with an increased kinase activity observed for this mutant variant and point to a possible explanation for its clinical sensitivity towards firstgeneration TKIs.38 Of interest, Gly719 mutations have been described as exhibiting resistance towards osimertinib,66 probably because of the neighboring Leu718 side chain,67 which makes important contributions for third-generation inhibitor binding.68 Osimertinib might be incompatible with an opened binding cleft upon G719S/A, acting similar to the L718V mutation, as outlined below. A mutation of the last glycine within the G-rich loop, G724S, is increasingly frequently observed.69,

70

Fassunke et al. revisited a cluster of 30 patients who were treated with third-

generation inhibitors and observed the emergence of G724S in conjunction with deletion mutations in exon 19 in four patients.51 It was speculated that similar to the EGFR crystal structure harboring the exon 20 insertion mutation,56 substitution with serine within the glycine-rich loop could induce a conformation resulting in a narrower binding site. In this way, third-generation inhibitors might experience steric repulsion, hindering their efficient binding. A similar effect of these mutations was assumed because the glycine-rich loop, ELREA motif, and helix C compose a network of regulatory elements, as outlined previously. This hypothesis was supported in MD simulations (Figure 4), which verified an enhanced flexibility of this network despite the strain introduced by


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deleting the ELREA sequence. Another valid conclusion might therefore include a loss of crucial interactions with third-generation TKIs, similar to G719S/C/A or L718V mutations. However, the study comprised a comprehensive profiling of 32 described inhibitors, including EGFR inhibitors of all three generations. In line with the findings from structural analyses, second-generation inhibitors, such as afatinib, are well tolerated because their quinazoline core avoids potential steric conflicts and their covalent mode of action provides sustained binding.51 These findings were validated and further elaborated by Brown et al., who found a G724S-induced conformation of the G-rich loop provoking a loss of interaction with osimertinib. Here, the observed resistance was attributed to disruption of the ligand’s interaction with Phe723, whereas second-generation inhibitors do not depend on this interaction and remained sensitive. Of interest, the study revealed the co-occurring exon 19 deletion mutation to be essential for osimertinib drug resistance, while the double mutation G724S+L858R was potently inhibited.71 Although insights into the structure of mutations within the glycine-rich loop were gained and sensitive inhibitors could be identified, further analyses are required to gain a deep understanding of the distinct effects. Particularly, a crystal structure of G724S-mutated EGFR is desired for clarification.


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Figure 4. Mutations within the flexible glycine-rich loop (GSGAFG motif) that is involved in ligand recognition. G719S or G724S mutations are thought to affect its dynamics, resulting in steric repulsion or loss of crucial interactions, involving Leu718 or Phe723 for example, with the binding of distinct inhibitors (gray surfaces). Alignment of EGFR crystal structures harboring an exon 20 insertion mutation (gray; PDB ID: 4LRM) and G719S (blue; PDB ID: 2ITO) as well as an MD simulation of EGFR-G724S (yellow).

Point Mutations Affecting the Binding Site Mediate Acquired Drug Resistance The efficacy of first- and second-generation TKIs such as gefitinib, or afatinib is limited by the emergence of a drug-resistance mutation of the gatekeeper residue Thr790 in exon 20.72, 73 The threonine in the back of the ATP-binding cleft is substituted with a sterically more demanding methionine side chain (T790M), which results in steric repulsion with the aniline moieties of quinazoline-based inhibitors (Figure 5A).74, 75 In addition, it was reported that the point mutation L858R activates the kinase accompanied by a decrease in affinity for the cofactor ATP. This fact allows for more efficient inhibition of this mutant with TKIs as compared to wild-type EGFR. The T790M mutation, on the other hand, restores the ATP affinity to a similar level as observed with WT-EGFR and thus mediates drug resistance.47 To overcome this type of drug resistance, a third generation of TKIs was developed. These TKIs have novel scaffolds, avoid steric interference with Met790, and take advantage of alkylating Cys797 while being selective over EGFR wild-type.26 The development of a novel point mutation C797S is therefore inevitable and again limits the success of these inhibitors. Substitution of the reactive cysteine by a less nucleophilic serine side chain prevents covalent bond formation, resulting in a loss of efficacy.76, 77 The effect of the gatekeeper mutation was clearly resolved


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through several available crystal structures of inhibitors based on quinazoline and related scaffolds. Furthermore, a 3D model of C797S-mediated resistance can be assessed through an existing cocrystal structure of WZ4003, the reversible counterpart of the third-generation inhibitor WZ4002,78,

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and a crystal structure of C797S-mutated EGFR bound with a derivative of

staurosporine,80, 81 Gö6976.82 It is believed, that WZ4003 somehow resembles the binding mode of covalent pyrimidine-based inhibitors upon mutation of the reactive cysteine (Figure 5B).68 The knowledge gained about the binding mode of TKIs within the EGFR binding site could be easily applied to additional point mutations affecting the binding site and that mediate drug resistance towards small-molecule inhibitors. Recent reports have identified mutations replacing the glycine at codon 796 in the EGFR with polar and sterically more demanding amino acids (G796S/R/D).66,

83-85

Revision of available crystal structures revealed a tremendous impact on

ligand binding. Because Gly796 is located at the lip of the active site, an enlarged side chain was found to cause steric interference with the solubilizing groups and core structures of available TKIs (Figure 5C). Another point mutation was observed in NSCLC patients. It affects the binding site at the hinge region, connecting the N- and C-lobes of the kinase domain. In this way, Leu792 is replaced by different side chains, with case reports mentioning mutations to His, Tyr, Phe, Arg, Pro and Val.66,

83

Leu792 has important interactions with a methoxy group included in third-

generation inhibitors rociletinib or osimertinib, which thus exhibits resistance towards replacement of the important amino acid (Figure 5C). Given these structural considerations, inhibitors that do not interact with Leu792 are believed to show a markedly reduced degree of resistance upon this type of mutation. A similar effect was observed for mutations in Leu718.66, 86-88 This particular side chain is part of a hydrophobic clamp motif that has important hydrophobic interactions with third-generation TKIs.68 Therefore, L718V results in a loss of ligand contacts, while L718Q results


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in steric conflict with rociletinib or osimertinib (Figure 5D). In addition, both substitutions found in lung cancer patients lead to a loss of hydrophobicity in close proximity to TKIs. However, the L718V mutation was found to retain sensitivity towards afatinib.66 Altogether, a detailed understanding of the distinct effects of mutations within the binding site has been established and will stimulate rational drug-design approaches for developing novel chemical entities that can overcome drug resistance in cancer patients with EGFR mutations.

Figure 5. Point mutations in the binding site directly interfere with ligand binding. (A) The T790M mutation hinders 1st- and 2nd-generation EGFR inhibitor binding through increased steric demand. Afatinib binding to wild-type EGFR (gray; PDB ID: 4G5J) aligned with T790M mutant apo-EGFR (blue; PDB ID: 3UG1). (B) The C797S mutation hinders 3rd-generation covalent inhibitor binding through reduced reactivity of the Ser797 side chain. Binding mode of the reversible analogue WZ4003 (gray; PDB ID: 5X2K) aligned with C797S-mutated EGFR (blue; PDB ID: 5XGN) as compared to the binding mode of covalent WZ4002 (black outlined; PDB ID: 3IKA). (C) G796R and L792H mutations interfere with 3rd-generation inhibitor binding, illustrated using the crystal structure of rociletinib (PDB ID: 5XDK). (D) The L718Q mutation interferes with 3rd-generation inhibitor binding (PDB ID: 5XDK).


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Summary and Outlook The field of protein structure determination has undergone a massive expansion over the last decade. Tools for the determination of high-resolution protein structures via X-ray crystallography have become more powerful and provide insights into the functions of numerous biological processes.89, 90 The ability to gain co-crystal structures of the target protein and small-molecule ligands supports the development of new drugs by promoting detailed understanding of the structure–activity relationship. However, successful structure determination by X-ray crystallography requires overcoming critical bottlenecks, which include gene design, protein expression and purification, crystallization, X-ray diffraction pattern acquisition, and finally, data set processing to yield the desired 3D model according to the electron density maps. Specifically, kinases often require more complex expression systems, such as insect cells, and successful crystallization is likely to depend on the protein sequence, taking intensive and time-consuming optimization cycles. Moreover, access to highly sophisticated equipment is required, essentially a high-energy synchrotron light source.91 Of note, the resulting structure typically provides a static snapshot of the protein, which is not necessarily the predominant state in biological systems.91, 92 Despite these and other limitations, protein crystallography is the method of choice for structure determination and accounted for 90% of the structures deposited in the PDB in 2016.93 The remaining 10% were solved by nuclear magnetic resonance (NMR) spectroscopy, which offers structure elucidation in solution and provides an alternative for proteins that are unlikely to crystallize. Moreover, real-time NMR provides information about the protein dynamics over a long period using exchange spectroscopy or relaxation measurements. An important feature during drug discovery is the mapping of chemical shifts, resulting in information about detailed protein–ligand


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interaction patterns. NMR studies have been limited to small proteins or fragments but thanks to technical advances in the field it can now be successfully applied to larger proteins.94, 95 Because these methodologies are experimentally intensive, time-consuming and have limitations, in silico modeling provides additional tools for accessing structural information. Among these, homology modeling has gained increasing interest because of the gap between the number of available protein sequences and disclosed protein structures. The method is based on the conditional evolutionary similarity of protein sequences, which results in similar threedimensional architectures within a protein family. Therefore, the known structure of one member allows a structural description of another family member by mapping the sequence of the target protein onto the resolved structure. Although homology modeling is less accurate than experimental methods, it is useful for hypotheses regarding overall protein folding or localization of ligand binding sites.96 Another method for investigating dynamic structural changes of side chains or even domain movements within the proteins is offered by MD simulations. With this approach, protein conformation transitions that are relevant to molecular function can be visualized. These investigations are based on crystal structures, which in turn illustrates the importance of the protein crystallography methodology.92 The technology of cryogenic electron microscopy (cryo-EM) was recognized with the 2017 Nobel Prize in Chemistry – which represents the impact to the field – and constitutes another method for high-resolution structure determination, applied to large proteins and protein complexes (>500 kDa).91, 97 It provides access to structures in solution or to membrane-spanning proteins that cannot be clarified by NMR or X-ray crystallography.91 Cryo-EM is currently not suited for high-throughput application because of intensive sample preparation, image capturing, and processing that require deep experience. However, the method allows for exact fitting of side


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chains and exact positioning of small molecules. It will thus certainly take a key role in drug discovery soon.97 In summary, the repertoire of 3D structure determination methodologies facilitates medicinal chemistry approaches, for example, in the field of kinase-targeted therapy of NSCLC, which we reviewed here. Specifically, we presented important activating and drug-resistant mutations of EGFR and Her2 and their effect on protein structures. We highlighted (i) a common mechanism of drug sensitivity upon mutations destabilizing a hydrophobic cluster, which is crucial for maintaining an inactive helix C-out conformation; (ii) the effect of deletion mutations in exon 19, which influence the regulatory helix C’s flexibility; and (iii) mutations within the glycine-rich loop, which affect ligand binding affinity by steric repulsion or loss of crucial interactions but are not yet fully understood. Moreover, we pointed out that (iv) the field lacks detailed insights into drug-resistant exon 20 insertion mutations in EGFR and Her2, but that (v) drug resistance arising from steric interference with active site-directed small-molecule inhibitors can be easily assessed using existing crystal structures. We are therefore certain that elusive mutants of EGFR and Her2 will be pinned down through further structural characterizations in the future. These findings will be directly relevant to structure-based drug design approaches and improve the efficiency of precision medicines. Ultimately, protein structure elucidations will push us further along the path to improved survival and quality of life for patients suffering from lung cancer.


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AUTHOR INFORMATION Corresponding Author *eMail: [email protected] (J.L.), [email protected] (D.R.). Phone: +49 (0)231/755-7080. Twitter: @DDHDortmund. Web: DDHDortmund.de. Present Addresses †PearlRiver Bio GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany (J.L.). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡J.N. and J.H. contributed equally. Notes J.L. is shareholder and full-time employee of PearlRiver Bio GmbH; D.R. is shareholder and consultant of PearlRiver Bio GmbH. ABBREVIATIONS cryo-EM, cryogenic electron microscopy; EGFR, epidermal growth factor receptor; MD, molecular dynamics; NMR, nuclear magnetic resonance; NSCLC, non-small cell lung cancer; PDB, protein data bank; TKI, tyrosine kinase inhibitor;.


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BIOGRAPHIES Janina Niggenaber studied Chemical Biology at TU Dortmund University (Germany). She joined the group of Daniel Rauh for her Bachelor studies on the biochemical evaluation of the detoxification enzyme Keap1. During her master thesis and following PhD studies she is engaged in the optimization of the expression and purification of clinically relevant mutants of the ErbB family with a strong focus on the crystallization of these mutants. Moreover, she is responsible for the cellular evaluation of inhibitors developed in the workgroup. Julia Hardick studied Chemical Biology at TU Dortmund University (Germany). She joined the group of Daniel Rauh for her Bachelor and Master studies dealing with structure-based design, synthesis and biochemical evaluation of p38α kinase inhibitors. In her current PhD studies, she focusses on the development of covalent inhibitors to target cancer-related drug targets, such as members of the ErbB-family, as well as the in vitro pharmacokinetic properties evaluation of these inhibitors. Jonas Lategahn studied Chemical Biology at TU Dortmund University (Germany) and performed his Bachelor studies at the Max Planck Institute of Molecular Physiology in the group of Andrey Antonchick. He joined the group of Daniel Rauh for his Master thesis followed by PhD studies in the field of Medicinal Chemistry with ErbB-driven non-small cell lung cancer as a key subject. During his PostDoctoral research at the Drug Discovery Hub Dortmund (DDHD), he focusses on the development of inhibitors of aberrant kinase signaling and acquired drug resistance. Daniel Rauh is a pharmacist by training and completed his PhD in the group of Gerhard Klebe in Marburg in 2003. After postdoctoral stays with Milton Stubbs in Halle and Kevan Shokat in San Francisco, he started his independent career at the Chemical Genomics Centre of the Max Planck


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Society in Dortmund. Since 2013 he serves as Professor and Chair of Chemical Biology and Medicinal Chemistry at TU Dortmund University. His research interests focus on the structurebased design and synthesis of small molecules for the modulation of biological systems. He is a co-founder of the Zentrum für Integrierte Wirkstoffforschung (ZIW) at TU Dortmund University and coordinates the Drug Discovery Hub Dortmund (DDHD), which aim at translating basic academic research into pharmaceutical application. REFERENCES (1) Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394-424.

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Table of content figure:


51


Figure 1. Inactive and active kinase domain conformations. (A) The inactive conformation (bound with lapatinib; PDB ID: 1XKK) exhibits an outwardly rotated helix αC (red) and a small helical turn within the activation loop (orange). It is in an equilibrium with the active conformation (bound with erlotinib; PDB ID: 1M17), which exhibits an inwardly rotated helix αC (green) that allows for a salt bridge between the catalytic lysine Lys745 and Glu762. Moreover, it is characterized by an extended activation loop (blue). (B) The side chain of Leu858 within the helical turn (orange) plays a crucial role in stabilizing the inactive conformation by packing into a hydrophobic cavity between helix αC (red) and glycine-rich loop (gray). Mutation of this amino acid to the polar Arg858 (PDB ID: 2ITT) therefore results in an equilibrium shift toward the active conformation. The QR-codes provide an augmented reality view of a corresponding 3D model.46 (C) Chemical structures of representative examples of the three TKI-generations of EGFR. 177x167mm (300 x 300 DPI)


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Figure 2. Deletion mutation of the ELREA motif in exon 19. MD simulations (blue) suggest that these mutations lock the kinase in its active conformation (green; PDB ID: 1M17) by restricting the flexibility of helix αC, which is crucial for adopting the inactive conformation (black outlined; PDB ID: 2JIV). 83x61mm (300 x 300 DPI)



Figure 3. Insertion mutations in exon 20. (A) Overview of the clinically most prevalent EGFR insertion mutations in exon 20 (PDB ID: 1M17). (B) A crystal structure of EGFR-D770_N771insNPG (blue; PDB ID: 4LRM) revealed a small wedge that locks the kinase in its active conformation by hindering Arg776 to stabilize the inactive conformation (red; PDB ID: 1XKK). (C) Overview of the clinically most prevalent Her2 insertion mutations in exon 20 (PDB ID: 3PP0). 177x119mm (300 x 300 DPI)


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Figure 4. Mutations within the flexible glycine-rich loop (GSGAFG motif) that is involved in ligand recognition. G719S or G724S mutations are thought to affect its dynamics, resulting in steric repulsion or loss of crucial interactions, involving Leu718 or Phe723 for example, with the binding of distinct inhibitors (gray surfaces). Alignment of EGFR crystal structures harboring an exon 20 insertion mutation (gray; PDB ID: 4LRM) and G719S (blue; PDB ID: 2ITO) as well as an MD simulation of EGFR-G724S (yellow). 83x58mm (300 x 300 DPI)



Figure 5. Point mutations in the binding site directly interfere with ligand binding. (A) The T790M mutation hinders 1st- and 2nd-generation EGFR inhibitor binding through increased steric demand. Afatinib binding to wild-type EGFR (gray; PDB ID: 4G5J) aligned with T790M mutant apo-EGFR (blue; PDB ID: 3UG1). (B) The C797S mutation hinders 3rd-generation covalent inhibitor binding through reduced reactivity of the Ser797 side chain. Binding mode of the reversible analogue WZ4003 (gray; PDB ID: 5X2K) aligned with C797Smutated EGFR (blue; PDB ID: 5XGN) as compared to the binding mode of covalent WZ4002 (black outlined; PDB ID: 3IKA). (C) G796R and L792H mutations interfere with 3rd-generation inhibitor binding, illustrated using the crystal structure of rociletinib (PDB ID: 5XDK). (D) The L718Q mutation interferes with 3rdgeneration inhibitor binding (PDB ID: 5XDK). 177x58mm (300 x 300 DPI)


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TOC Figure 55x55mm (300 x 300 DPI)


Structure Defines Function â Clinically Relevant ... - ACS Publications

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