Discovery of Novel eEF2K Inhibitors Using HTS Fingerprint Generated from Predicted Profiling of Compound-Protein Interactions

Processing Peptidase

Τα είδη του γένους Leishmania αποτελούν υποχρεωτικά ενδοκυττάρια πρωτοζωικά παράσιτα που προκαλούν ένα ευρύ φάσμα ασθενειών, τις λεισμανιάσεις. Η λεϊσμανίαση θεωρείται νόσος εξέχουσας σπουδαιότητας, με 2 εκατομμύρια νέα κρούσματα το χρόνο, χρήζουσας μεγάλης κοινωνικής και οικονομικής σημασίας. Για τον έλεγχο των συνεχώς αυξανόμενων κρουσμάτων, είναι επιτακτική ανάγκη η ανάπτυξη νέων μη-τοξικών φαρμάκων που θα στοχεύουν σε μόρια-στόχους σημαντικά για την ολοκλήρωση του παρασιτικού κύκλου ζωής. Κατ’ αυτόν τον τρόπο κρίνεται απαραίτητη η μελέτη μηχανισμών και παραγόντων μολυσματικότητας του παρασίτου, που ελέγχουν τον κυτταρικό κύκλο και τη διαφοροποίηση του παρασίτου. Στα πλαίσια αυτά, έχει δειχθεί ότι η επισωμική υπερέκφραση της συνδετικής ιστόνης Η1 του παρασίτου Leishmania (LeishH1) οδηγεί στην καθυστέρηση της ολοκλήρωσης του κυτταρικού κύκλου, αλλά παράλληλα και στη μείωση του ρυθμού διαφοροποίησης των παρασίτων από προμαστιγωτές σε αμαστιγωτές μορφές, έχοντας σαν αποτέλεσμα τη μείωση της μολυσματικότητας του παρασίτου τόσο in vitro όσο και in vivo. Στόχος αυτής της διατριβής ήταν η ανάδειξη μορίων που επηρεάζουν τη μολυσματικότητα του παρασίτου, μελετώντας το προτέωμα των μη μολυσματικών παρασίτων που υπερεκφράζουν την LeishH1. Η συγκριτική μελέτη των παρασίτων που υπερεκφράζουν τη LeishH1 σε σχέση με τα παράσιτα ελέγχου, είχε επίσης ως σκοπό την περαιτέρω μελέτη του βιολογικού ρόλου της LeishH1 στα παράσιτα και τη διερεύνηση του ρόλου της στη γονιδιακή ρύθμιση του παρασίτου. Η συγκριτική πρωτεομική ανάλυση με ηλεκτροφόρηση δυο-διαστάσεων, των παρασίτων που υπερεκφράζουν την LeishH1 σε σχέση με τα παράσιτα ελέγχου, κατέδειξε πως μόνο μια μικρή ομάδα πρωτεϊνών παρουσιάζει διαφορική έκφραση. Συγκεκριμένα, τρεις πρωτεΐνες [heat shock protein 83 (HSP83), eukaryotic elongation factor 2(eEF-2), alpha subunit of the mitochondrial processing peptidase (α-MPP)] παρουσιάζουν χαμηλότερα επίπεδα έκφρασης ενώ άλλες δυο (α/β τουμπουλίνη, ΜΑΡ) παρουσιάζουν μεγαλύτερη κατανομή έκφρασης. Πειράματα αλυσιδωτής αντίδρασης πολυμεράσης αντίστροφης μεταγραφάσης πραγματικού χρόνου, επιβεβαίωσαν το αποτέλεσμα αυτό, υποδηλώνοντας ότι η LeishH1 δεν είναι γενικός καταστολέας της μεταγραφής αλλά επηρεάζει ένα ειδικό υποσύνολο πρωτεϊνών, σε προ- ή μετα-μεταγραφικό επίπεδο. Ανάμεσα στις πρωτεΐνες με διαφορική έκφραση ήταν και η τουμπουλίνη. Η διαφορική έκφραση της πρωτεΐνης αντικατοπτρίζεται άμεσα στη μορφολογία των παρασίτων που υπερεκφράζουν την LeishH1, καθώς τα παράσιτα αυτά παρουσιάζουν μικρότερο και πιο στρογγυλό σχήμα και μεγαλύτερη μορφολογική ετερογένεια. Μια ακόμα πρωτεΐνη με διαφορική έκφραση, ήταν και η HSP83, η οποία παρουσίασε χαμηλότερα επίπεδα. Θέλοντας να εξακριβώσουμε σε ποιο επίπεδο παρεμβαίνει η LeishH1 στο μηχανισμό έκφρασης της HSP83, συγκρίναμε τα επίπεδα του mRNA και η ανάλυση κατέδειξε ότι δεν υπάρχουν διαφορές στα επίπεδα αυτά. Επίσης, μελετήθηκε ο ρυθμός έκφρασης της πρωτεΐνης μέσω της μεταβολικής σήμανσης των πρωτεϊνών και αποκαλύφθηκε πως σε αυτό το στάδιο εντοπίζεται η παρεμβολή της LeishH1, καθώς τα παράσιτα που την υπερεκφράζουν παρουσιάζουν χαμηλότερο ρυθμό έκφρασης της HSP83. Συμπερασματικά διαφαίνεται πως υπάρχει μια συσχέτιση μεταξύ μονοπατιών που εμπλέκονται στην αντίσταση έναντι φαρμακευτικών ουσιών, στην απόπτωση και τη μολυσματικότητα. Κατ’ αυτόν τον τρόπο, τα ειδικά σήματα και οι μηχανισμοί που ρυθμίζουν τη διαφοροποίηση/μολυσματικότητα του παρασίτου και την απόπτωση/απόκριση στο στρες, χρήζουν περαιτέρω διερεύνησης καθώς φαίνεται να αποτελούν τις δυο όψεις του ίδιου νομίσματος.

Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

Biochemical Journal ◽

10.1042/bj20111530 ◽

2012 ◽

Vol 442 (3) ◽

pp. 681-692 ◽

Cited By ~ 37

Author(s):

Sébastien Pyr Dit Ruys ◽

Xuemin Wang ◽

Ewan M. Smith ◽

Gaëtan Herinckx ◽

Nusrat Hussain ◽

...

Keyword(s):

Catalytic Domain ◽

Elongation Factor ◽

Translation Elongation ◽

Intact Cells ◽

Elongation Factor 2 ◽

293 Cells ◽

A Site

eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases.

In vitro interaction of eukaryotic elongation factor 2 with synthetic oligoribonucleotide that mimics GTPase domain of rat 28S ribosomal RNA

The International Journal of Biochemistry & Cell Biology ◽

10.1016/s1357-2725(01)00122-4 ◽

2002 ◽

Vol 34 (3) ◽

pp. 263-268

Author(s):

Wen-jun He ◽

Shuang Tang ◽

Wang-yi Liu

Keyword(s):

Ribosomal Rna ◽

Elongation Factor ◽

Gtpase Domain ◽

Elongation Factor 2 ◽

In Vitro Interaction

A high-throughput screening assay for eukaryotic elongation factor 2 kinase inhibitors

Acta Pharmaceutica Sinica B ◽

10.1016/j.apsb.2016.04.002 ◽

2016 ◽

Vol 6 (6) ◽

pp. 557-563 ◽

Cited By ~ 6

Author(s):

Ting Xiao ◽

Rui Liu ◽

Christopher G. Proud ◽

Ming-Wei Wang

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Kinase Inhibitors ◽

Elongation Factor ◽

Screening Assay ◽

Elongation Factor 2 ◽

High Throughput Screening Assay

Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy

International Journal of Molecular Sciences ◽

10.3390/ijms22052408 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2408

Author(s):

Bin Zhang ◽

Jiamei Zou ◽

Qiting Zhang ◽

Ze Wang ◽

Ning Wang ◽

...

Keyword(s):

Cancer Cells ◽

Elongation Factor ◽

Cancer Drug ◽

Discovery Research ◽

Elongation Factor 2 ◽

Drug Discovery Research ◽

Anti Cancer

Eukaryotic elongation factor 2 kinase (eEF2K or Ca2+/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient depletion, and acidosis. The physiological function of eEF2K and its role in the development and progression of cancer are here reviewed in detail. In addition, a summary of progress for in vitro eEF2K inhibitors from anti-cancer drug discovery research in recent years, along with their structure–activity relationships (SARs) and synthetic routes or natural sources, is also described. Special attention is given to those inhibitors that have been already validated in vivo, with the overall aim to provide reference context for the further development of new first-in-class anti-cancer drugs that target eEF2K.

Novel Inhibitors of Eukaryotic Elongation Factor 2 Kinase: In Silico, Synthesis, and in Vitro Studies

10.26434/chemrxiv.13299149.v1 ◽

2020 ◽

Author(s):

Ferah CÖMERT ÖNDER ◽

Serdar DURDAĞI ◽

Nermin Kahraman ◽

Tuğçe Nur Uslu ◽

Hakan Kandemir ◽

...

Keyword(s):

Breast Cancer ◽

In Silico ◽

Covalent Binding ◽

Elongation Factor ◽

Elongation Factor 2 ◽

Pharmacologically Active ◽

Coumarin Ring

Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual alpha kinase whose expression is highly upregulated in various cancers and contributes to tumor growth, metastasis, and progression. More importantly, expression of eEF2K is associated with poor clinical outcome and shorter patient survival triple negative breast cancer (TNBC). Therefore, eEF2K is an emerging molecular target for development of novel targeted therapeutics and precision medicine in solid cancers. However, currently potent, and specific inhibitors of eEF2K are not available for clinical translation. In the current study, we investigated the effects of various newly designed and synthesized a series of compounds with coumarin scaffold substitutions in inhibiting eEF2K activity using in silico approaches and in vitro studies in TNBC cells. We utilized an amide substitution at 3-position on the coumarin ring with their pharmacologically active groups containing pyrrolidine, piperidine, morpholine and piperazine groups with –(CH2)2– bridged for aliphatic amides. To evaluate substituent effects on coumarin scaffold, boronic acid pinacol ester and boronic acids on phenyl rings were investigated using in silico and in vitro analyses. Due to their ability to form covalent binding to the target enzyme, we investigated the effects of boron containing groups on functionalized coumarin ring (3 compounds) and designed novel aliphatic and aromatic derivatives of coumarin scaffolds (10 compounds) and phenyl ring with boron groups (4 compounds). In silico analysis and molecular docking studies were performed using the Glide/SP module of Maestro molecular modeling package. According to obtained results, structure activity relationship (SAR) was carried out. Among the newly designed, synthesized, and tested compounds, our in vitro findings revealed that several compounds displayed a highly effective eEF2K inhibition at submicromolar concentration in in vitro breast cancer cells. In conclusion, we identified novel eEF2K inhibitors as promising anticancer drug substance candidates which should be further evaluated by in vivo studies, preclinical and clinical studies.

Novel Inhibitors of Eukaryotic Elongation Factor 2 Kinase: In silico, synthesis and in vitro studies

Bioorganic Chemistry ◽

10.1016/j.bioorg.2021.105296 ◽

2021 ◽

pp. 105296

Author(s):

Ferah Comert Onder ◽

Serdar Durdagi ◽

Nermin Kahraman ◽

Tugce Nur Uslu ◽

Hakan Kandemir ◽

...

Keyword(s):

In Silico ◽

Elongation Factor ◽

In Vitro Studies ◽

Elongation Factor 2 ◽

Eukaryotic Elongation Factor

Novel Inhibitors of Eukaryotic Elongation Factor 2 Kinase: In Silico, Synthesis, and in Vitro Studies

10.26434/chemrxiv.13299149 ◽

2020 ◽

Author(s):

Ferah CÖMERT ÖNDER ◽

Serdar DURDAĞI ◽

Nermin Kahraman ◽

Tuğçe Nur Uslu ◽

Hakan Kandemir ◽

...

Keyword(s):

Breast Cancer ◽

In Silico ◽

Covalent Binding ◽

Elongation Factor ◽

Elongation Factor 2 ◽

Pharmacologically Active ◽

Coumarin Ring

Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual alpha kinase whose expression is highly upregulated in various cancers and contributes to tumor growth, metastasis, and progression. More importantly, expression of eEF2K is associated with poor clinical outcome and shorter patient survival triple negative breast cancer (TNBC). Therefore, eEF2K is an emerging molecular target for development of novel targeted therapeutics and precision medicine in solid cancers. However, currently potent, and specific inhibitors of eEF2K are not available for clinical translation. In the current study, we investigated the effects of various newly designed and synthesized a series of compounds with coumarin scaffold substitutions in inhibiting eEF2K activity using in silico approaches and in vitro studies in TNBC cells. We utilized an amide substitution at 3-position on the coumarin ring with their pharmacologically active groups containing pyrrolidine, piperidine, morpholine and piperazine groups with –(CH2)2– bridged for aliphatic amides. To evaluate substituent effects on coumarin scaffold, boronic acid pinacol ester and boronic acids on phenyl rings were investigated using in silico and in vitro analyses. Due to their ability to form covalent binding to the target enzyme, we investigated the effects of boron containing groups on functionalized coumarin ring (3 compounds) and designed novel aliphatic and aromatic derivatives of coumarin scaffolds (10 compounds) and phenyl ring with boron groups (4 compounds). In silico analysis and molecular docking studies were performed using the Glide/SP module of Maestro molecular modeling package. According to obtained results, structure activity relationship (SAR) was carried out. Among the newly designed, synthesized, and tested compounds, our in vitro findings revealed that several compounds displayed a highly effective eEF2K inhibition at submicromolar concentration in in vitro breast cancer cells. In conclusion, we identified novel eEF2K inhibitors as promising anticancer drug substance candidates which should be further evaluated by in vivo studies, preclinical and clinical studies.