Computational studies on KRAS inhibitors using web based tools

COMPUTATIONAL STUDIES ON KRAS INHIBITORS USING WEB BASED TOOLS T abajo de Fin de Más e Ai o Díaz Oli a Tu o izado po D . José M. Pad ón Más e en Biomedicina Julio 2024 2 La p esen e memo ia de in es igación ha sido ealizada po Ai o Díaz Oli a du an e el cu so académico 2023-2024 en el g upo BioLab del Ins i u o Uni e si a io de Bio- O gánica “An onio González” (IUBO-AG) y bajo la di ección del D . José M. Pad ón. El abajo o ma pa e de la línea de in es igación Diseño, descub imien o y e aluación de á macos an icance ígenos. La Laguna, en la echa de i ma al pie. 3 TABLE OF CONTENTS ABSTRACT 4 RESUMEN 4 INTRODUCTION 5 HYPOTHESIS 11 OBJECTIVES 11 MATERIALS AND METHODS 12 RESULTS & DISCUSSION 17 CONCLUSIONS 33 REFERENCES 34 4 ABSTRACT In his wo k, a compu a ional s udy o he KRAS p o ein wi h wo o i s inhibi o s o he G12C a ian , so o asib and adag asib, has been ca ied ou . Ini ially, he in e ac ion be ween he KRAS G12C inhibi o s and he p o ein was s udied using publicly a ailable so wa e (Swissdock). Nex he in e ac ions we e s udied o he G12D, G12R and G13C a ian s o KRAS. In a e o o compa e he esul s ob ained wi h hose gi en by p o essional so wa e, hese same in e ac ions we e s udied using licensed so wa e MOE. All he esul s ob ained om he di e en so wa e and he esul s ob ained om he di e en KRAS a ian s we e compa ed. RESUMEN En es e abajo se ha ealizado un es udio compu acional de la p o eína KRAS con dos de sus inhibido es pa a la a ian e G12C, so o asib y adag asib. Inicialmen e, se es udió la in e acción en e los inhibido es de KRAS G12C y la p o eína u ilizando un so wa e disponible públicamen e (Swissdock). A con inuación, se es udia on las in e acciones pa a las a ian es G12D, G12R y G13C de KRAS. En un es ue zo po compa a los esul ados ob enidos con los p opo cionados po el so wa e p o esional, es as mismas in e acciones se es udia on u ilizando el so wa e con licencia MOE. Se compa a on odos los esul ados ob enidos con los dis in os p og amas in o má icos y los esul ados ob enidos con las dis in as a ian es de KRAS. INTRODUCTION 5 INTRODUCTION Nowadays, he numbe o cance cases wo ldwide has inc eased signi ican ly due o he inc ease in li e expec ancy and cu en en i onmen al condi ions. Acco ding o a s udy by he INE, in Spain in 2022, cance mo ali y became he second cause o dea h jus behind ca dio ascula diseases [1]. Among he a ious ypes o cance ha exis , i is impo an o ake in o accoun hose ha cause he g ea es mo ali y in he popula ion. Fo women hey a e lung, b eas , colo ec al and panc ea ic cance , while o men hey a e lung, p os a e, colo ec al, panc ea ic and li e cance [2]. O he deadlies cance s men ioned abo e, we will ocus on panc ea ic cance , whe e he main isks a e ad anced age, ace, gende and en i onmen al ac o s such as smoking, die , e c. In ela ion o molecula ac o s, he mos equen umo supp esso genes ha a e inac i a ed a e P16 in 95%, P53 in up o 75%, DPC4 in 50% and BRCA2. The mos equen gene ic al e a ion o oncogenes is he KRAS gene, ound in 80% o 100% o cases [3-4]. The KRAS p o ein ( igu e 1) is an impo an oncogene ha plays a key ole in he de elopmen and p og ession o a ious cance s. The KRAS gene codes o a GTPase p o ein ha is pa o he in acellula RAS/MAPK signaling pa hway, which egula es cellula p ocesses such as p oli e a ion, di e en ia ion and apop osis. The mos common mu a ions a e ound in codons 12, 13, 61 and 146 o he KRAS gene. Mu a ed KRAS is he main oncogenic d i e o panc ea ic duc al adenoca cinoma (PDAC) and an a ac i e ea men a ge . To da e, no e ec i e ea men s ha e been iden i ied o pa ien s wi h KRAS-mu a ed PDAC, excep o he unusual KRAS G12C mu a ions [4-8]. INTRODUCTION 6 Figu e 1. S uc u e o wild ype human GTP KRAS p o ein. In cance s caused by mu an KRAS, he p o ein is blocked in he ac i e GTP-bound s a e cons i u i ely, h ough a de ec in he swi ch-o mechanism. As such, he mu an p o ein esembles he no mal KRAS p o ein om a s uc u al pe spec i e, making he apeu ic a ge ing ex emely challenging. KRAS is a membe o a la ge amily o ela ed p o eins, which sha e e y simila GDP/GTP binding domains, making a ge ed he apies di icul . The KRAS G12C mu a ion (Figu e 2) is cha ac e ized by an amino acid subs i u ion o cys eine in he GTP-binding si e o he KRAS p o ein, leading o cons an ac i a ion o he RAS/MAPK signaling pa hway and uncon olled cell p oli e a ion and su i al [9]. Figu e 2. S uc u e o mu an KRAS G12C. The inhibi o s ha ha e shown o be e ec i e agains he KRAS G12C mu an a e so o asib (also known as AMG 510 o Lumak as) and adag asib (also known as MRTX849). These di ec inhibi o s ac by selec i ely o ming a co alen bond wi h Cys 12 esidue wi hin he swi ch II pocke o he KRAS G12C p o ein, hus blocking KRAS in an inac i e INTRODUCTION 7 s a e o s op cell p oli e a ion (Figu e 3). P eclinical s udies showed ha so o asib and adag asib selec i ely a ec he iabili y o KRAS G12C mu an cell lines, bu do no a ec cell lines wi h o he KRAS mu a ions in i o and in i o [10]. Figu e 3. a) KRAS has GTPase ac i i y and can con e GTP in o GDP by hyd olyzing he gamma phospha e on GTP. The inac i e and ac i e s a es o KRAS a e egula ed by GAP and GEF, espec i ely. RTKs a e he second majo ype o cell su ace ecep o s wi h a wide ange o unc ions, including p omo ing KRAS ac i a ion and subsequen mul iple e ec o pa hways, especially he RAF-MEK-ERK and PI3K-AKT-mTOR pa hways. b) Compa ed wi h wild- ype KRAS, which main ains a balance be ween inac i e and ac i a ed s a es, Cys 12 (C12) mu a ions des oys GTPase ac i i y o KRAS and locks in he GTP-bound s a e. In con as , he small molecule d ug so o asib and adag asib can o m a co alen bond wi h C12 in he KRAS G12C p o ein, causing KRAS o be in an inac i e s a e. So o asib So o asib (AMG510, Figu e 4) is a py idopy imidine ha has a py ido[2,3-d]py imidin- 2(1H)-one subs i u ed wi h 4-me hyl-2-(p opan-2-yl)py idin-3-yl, (2S)-2-me hyl -4-(p op-2- INTRODUCTION 8 enoyl)pipe azin-1-yl, luo o and 2- luo o-6-hyd oxyphenyl g oups a posi ions 1, 4, 6 and 7, espec i ely. I is app o ed o he ea men o pa ien s wi h lung cance and o he non-small cell solid cance s ha ha e KRAS G12C mu a ions. I has a ole as an an ineoplas ic agen . I is a membe o ac ylamides, an N-acylpipe azine, a py idopy imidine, a membe o mono luo obenzenes, a membe o me hylpy idines, a e ia y ca boxamide, a e ia y amino compound and a membe o phenols. In e ms o physicochemical p ope ies, i has a molecula weigh o 560.6 g/mol, a solubili y o 0.03 mg/mL a pH 6.8 and a pKa o 4.56 and 8.56. Figu e 4. Molecula s uc u e o so o asib. Fo abso p ion o a once-daily dose o 960 mg o so o asib, i achie es a C max o 7.50 µg/mL, wi h a median T max o 2.0 hou s and an AUC 0-24 h o 65.3 h *μg/mL. So o asib is 74% exc e ed in he eces and 6% in he u ine. 53% o he dose eco e ed in eces and 1% o he dose eco e ed in u ine a e in he unchanged pa en compound o m. Typically, GTP binds o KRAS, ac i a ing he p o ein and p omo ing e ec o s o he MAP kinase pa hway. GTP is hyd olyzed o GDP and KRAS is inac i a ed (Figu e 5). KRAS G12C mu a ions al e GTP hyd olysis, lea ing i in an ac i e o m. So o asib binds o he Cys 12 esidue in KRAS G12C mu an, keeping he p o ein in i s inac i e o m. The cys eine esidue a ge ed by so o asib is no p esen in wild- ype KRAS, p e en ing o - a ge e ec s. This mu a ion is p esen in 13% o non-small cell lung cance s, 3% o colo ec al and appendiceal cance s and 1-3% o solid umo s [11]. INTRODUCTION 9 Figu e 5. Binding pose o so o asib o he KRAS-G12C p o ein. O ange: Swi ch II domain; Yellow: esidual C12; Blue: a c yp ic pocke composed o H95, Y96 and Q99. So o asib (g een) and GDP (g ey) a e shown in s icks. Adag asib Adag asib (Figu e 6) is ano he d ug ha ac s as a KRAS G12C p o ein inhibi o simila o so o asib. I has a molecula weigh o 604.1 g/mol. In e ms o abso p ion, he AUC and C max o adag asib inc ease dose-p opo ionally be ween 400 mg and 600 mg. The Tmax is app oxima ely 6 hou s. Fu he mo e, adag asib has a high o al bioa ailabili y and is able o pene a e he cen al ne ous sys em. I is elimina ed in he eces and u ine. In pa ien s ecei ing a single dose o adiolabelled adag asib, 75% o he dose was eco e ed in eces (14% unchanged), while 4.5% was eco e ed in u ine (2% unchanged). Adag asib is a small molecule inhibi o o he mu an KRAS G12C p o ein ound in up o 13% o e ac o y non-small cell lung cance cases. Ele a ions o se um amino ans e ases a e common du ing ea men wi h adag asib and a p opo ion o pa ien s de elop clinically appa en li e inju y ha can be se e e [12]. RESULTS & DISCUSSION 16 Figu e 11. Sc eensho om he new SwissDock websi e. RESULTS & DISCUSSION 17 RESULTS & DISCUSSION Today he e is a wide a ie y o cance s and umo s ha can mani es in any pa o he body and ac in a a ie y o ways om being ha mless o being e ociously le hal. W d ew ou a en ion o panc ea ic cance . The e a e se e al genes ha inc ease he isk o panc ea ic cance . These genes comp ise: • TP53 • CDKN2A • SMAD4/DPC4 • BRCA1 and BRCA2. • KRAS We ocused ou a en ion on he la e , as i is a p o ein o in e es o cell g ow h and di ision. A mu a ion in his p o ein leads o inc eased p oli e a ion o cance cells. Nex , we e ie ed in o ma ion on he p o ein in he PDB da abase o bo h he wild- ype and mu an p o ein. Wi hin he PDB we can ob ain in o ma ion o in e es on he p o ein such as name, yea o publica ion, au ho s who syn hesized i , me hod by which he cha ac e iza ion was ob ained, he o ganism om which i was ob ained and he ype o mac omolecule i belongs o (Figu e 12). Figu e 12. In o ma ion abou KRAS WT ob ained om PDB. In o de o be able o ca y ou he s udy, he esolu ion o he p o ein mus be aken in o accoun , gi en ha alues g ea e han 2 Å would ha e a poo esolu ion o would be hea y RESULTS & DISCUSSION 18 enough o ca y ou he s udy. On he o he hand, i was necessa y o make su e ha he p o ein belongs o he Homo sapiens o ganism. Fo he old e sion o SwissDock, i was necessa y o use he PDB in o de o download he desi ed p o ein in PDB o ma . Subsequen ly, he SMILE o ma s o he wo KRAS G12C inhibi o s so o asib and adag asib we e ob ained om Pubchem. Once he o ma s ha e been ob ained, OpenBabel so wa e was used o ans o m he SMILE o ma in o ano he ha can be ead by he ee SwissDock so wa e. This o ma is mol2. Subsequen ly, he PDB o ma o he p o ein and mol2 o he inhibi o was in oduced in o SwissDock. As o he docking ime, since i is a ee so wa e, i can ake om minu es o days. To a oid ha ing o wai , SwissDock gi es you he op ion o en e ing you email add ess and you will be no i ied when he docking is inished (Figu e 13). Figu e 13. Sc eensho om he SwissDock websi e o docking. RESULTS & DISCUSSION 19 Unluckily due o he so wa e upda e, i is cu en ly impossible o access he in o ma ion o he docking pe o med be o e he upda e, gi ing an e o and he impossibili y o access he esul s (Figu e 14). Figu e 14. Image o he e o when accessing he esul s a e docking. As o he new so wa e upda e, i has been designed in such a way ha i is mo e use - iendly. I is no longe necessa y o download he PDB iles om he p o ein, no is i necessa y o ans o m he SMILE o ma s o mol2. As o he p o ein- a ge in e ac ion, i is isualized in such a way ha ionic bonds, hyd ogen bonds, π-bonds, hyd ophobic con ac s can be obse ed and shows he p o ein su ace (Figu e 15). Figu e 15. Example o di e en ypes o p o ein-inhibi o in e ac ions (le ). Example o in e ac ion be ween p o ein su ace and inhibi o ( igh ). SwissDock is a ee so wa e and has some limi a ions such as he size o he p o ein. In addi ion, wi h he new e sion hey a oid docking o se e al days. Thus, i i is expec ed ha he esul is p olonged in ime, hey wa n ha i is no possible o pe o m he calcula ions. I can also lead o e o s when en e ing he desi ed p o ein (Figu e 16). RESULTS & DISCUSSION 20 Figu e 16. E o o he so wa e o ecognise he p o ein. So o asib As a o emen ioned, so o asib is a d ug ha inhibi s he KRAS G12C mu a ed p o ein. We un a compu a ional s udy be ween KRAS p o eins and so o asib. KRAS w I was decided o pe o m docking o he KRAS w p o ein oge he wi h he inhibi o in o de o ha e in o ma ion abou how he so wa e will ac agains a hypo he ical p o ein- inhibi ion in e ac ion. Al hough he esolu ion o he PDB s uc u e 6VC8 is la ge han 2Å (Figu e 17), he compu a ional s udy was ca ied ou due o he absence o be e op ions. RESULTS & DISCUSSION 21 Figu e 17. In o ma ion abou KRAS w . Figu e 18. In e ac ion be ween KRAS and so o asib. RESULTS & DISCUSSION 22 Figu e 19. Chemical in e ac ion be ween KRAS and so o asib. As shown in Figu es 18 and 19, so o asib binds on he ou side o he p o ein. In addi ion, he e a e hyd ogen bonding in e ac ions o he hyd oxyl g oup o so o asib wi h Lys 117 ( ed p o ein) and HSD 95 (whi e p o ein). In a compa ison wi h MOE so wa e (Figu e 20), Lys 117 binds o he py idine ni ogen. Figu e 20. Chemical in e ac ion be ween KRAS and so o asib using MOE. RESULTS & DISCUSSION 23 As expec ed, he docking simula ion o so o asib wi h KRAS w shows ha he d ug does no i in o he binding si e. KRAS G12C The in e ac ion be ween he KRAS G12C mu an (Figu e 21, PDB ID: 5V9O) and so o asib was modelled in o de o compa e he da a ob ained on he in e ac ion wi h hose epo ed in he li e a u e. Figu e 21. In o ma ion abou KRAS G12C mu an . Figu e 22. Snapsho s o he in e ac ion be ween KRAS G12C mu an and so o asib. As can be seen in he Figu es 22 & 23, he inhibi o is in oduced in o he p o ein (binding si e), p oducing he KRAS G12C-so o asib in e ac ion. The e is also in e ac ion be ween he allylic ke one and Gly 15. RESULTS & DISCUSSION 24 Figu e 23. P edic ed in e ac ion be ween KRAS G12C mu an and so o asib. Figu e 24 shows he in e ac ions o he inhibi o wi h he p o ein using MOE so wa e. I can be seen ha he p edic ed in e ac ions a e di e en be ween he Swissdock so wa e and MOE so wa e. Figu e 24. P edic ed in e ac ion be ween KRAS G12C mu an and so o asib using MOE RESULTS & DISCUSSION 25 KRAS G12R The KRAS G12R mu an (Figu e 25, PDB ID: 6CU6) has a simila s uc u e o G12C. Among he cha ac e is ics o 6CU6 we highligh ha i has an accep able esolu ion and belongs o he Homo sapiens amily. Figu e 25. In o ma ion abou KRAS G12R mu an . Figu e 26. P edic ed in e ac ion be ween KRAS G12R mu an and so o asib. RESULTS & DISCUSSION 32 Simila ly, he web pla o m did no accep he mol2 ile (Figu e 38). The e o e, we could no un he p oposed s udy. Figu e 38. Sc eensho o e o message when en e ing he mol2 ile o adag asib. CONCLUSIONS 33 CONCLUSIONS A e docking he p oposed inhibi o s agains he KRAS p o ein, he ollowing conclusions we e ob ained: I is obse ed ha he e a e sligh di e ences in he p edic ion o KRAS-so o asib in e ac ion depending on he so wa e used: Swissdock (public use so wa e) and MOE (comme cial use so wa e). Bo h sys ems p edic in e ac ions on di e en he e oa oms and di e en posi ions o he amino acids o he p o ein. Wi h Swissdock so wa e, i was obse ed ha he e is no in e ac ion ha is p omising o KRAS G12R and G12D. Howe e , simila i y is seen be ween G12C and G13C. The e o e in e ac ion s udies be ween KRAS G13C-so o asib and inhibi o should be ca ied ou o his mu an . Fo adag asib, i was no possible o pe o m docking s udies wi h Swissdock so wa e due o p oblems in en e ing he s uc u e in ei he SMILES and mol2 o ma o he inhibi o . Finally, he use o public so wa e such as Swissdock allows any s uden o pe o m s udies on he in e ac ions be ween p o eins and desi ed a ge molecules. Howe e , his so wa e has se e al d awbacks, such as he wai ing ime o ob ain he esul s, which can las se e al days, and e o s in he so wa e i sel (da a en y, da a sizes, los connec ions, e c.). Al hough i is ideal o a oid high usage o p i a e licenses, da a should be co obo a ed wi h p o essional so wa e ha is highly eliable. REFERENCES 34 REFERENCES [1] INE. Causas de mue e dos años después del comienzo de la pandemia. Re ie ed om: h ps://www.ine.es/ss/Sa elli e?L=es_ES&c=INECi asINE_C&cid=1259954008107& p=1254735116567&pagename=P oduc osYSe icios%2FINECi asINE_C%2FPYSD e alleCi asINE [2] B ay, F., La e sanne, M., Sung, H., Fe lay, J., Siegel, R. L., Soe joma a am, I., & Jemal, A. (2024). Global cance s a is ics 2022: GLOBOCAN es ima es o incidence and mo ali y wo ldwide o 36 cance s in 185 coun ies. CA: a cance jou nal o clinicians, 74(3), 229-263. [3] Pe ei a, A. G. M., Noal, L. B., de Cama go, L. E. A., Ia skiu, C. E. A., Viei a, J. B., Figuei edo, E. F., & Colaci e, J. (2022). A aliação epidemiológica do Cânce de pânc eas no B asil: mo alidade e a o es de isco: Epidemiological e alua ion o panc ea ic Cance in B azil: mo ali y and isk ac o s. B azilian Jou nal o De elopmen , 8(10), 68595-68603. [4] U eña, J. F. (2017). Cánce de pánc eas. Re is a Médica Sine gia, 2(8), 3-6. [5] McCo mick, F. (2016). KRAS p o ein as a d ug a ge . Jou nal o molecula medicine, 94, 253-258. [6] Luo, J. (2021, Feb ua y). KRAS mu a ion in panc ea ic cance . In Semina s in oncology (Vol. 48, No. 1, pp. 10-18). WB Saunde s. [7] Olmedo Pimen el, Y. (2015). Mu aciones en el Gen KRAS en mues a de ejidos umo ales (Doc o al disse a ion, Uni e sidad de Panamá. Vice ec o ía de In es igación y Pos g ado.). [8] S umbe g, D., Schul heis, B., Ebe , M.P., Si eke, J.T., Ke kho , A., Ho heinz, R., Beh inge , D., Schmid , W.E., Göke , E., Dosso, S.D., Kneba, M., Yalcin, S., O e kamp, F., Schlegel, F., Dommach, M., Roh be g, R., S einme z, T., Bach, F., Reu e , D., Ilyas, R.A., & Escoba , N.E. (2016). T ai emen de pa ien s diagnos iqués a ec un adénoca cinome canalai e du panc éas au moyen de l'u ilisa ion d'an ico ps monoclonaux con e le écep eu de ac eu de c oissance épide mique (eg ). [9] Liu, J., Kang, R., & Tang, D. (2022). The KRAS-G12C inhibi o : ac i i y and esis ance. Cance Gene The apy, 29(7), 875-878. [10] Se on-Roge s, S. (2020). KRAS-G12C in he c osshai s. Na u e Re iews Cance , 20(1), 3-3. REFERENCES 35 [11] Yang, A., Li, M., & Fang, M. (2021). The esea ch p og ess o di ec KRAS G12C mu a ion inhibi o s. Pa hology & Oncology Resea ch, 27, 631095. [12] Wu, F., Zhou, Y., Li, L., Shen, X., Chen, G., Wang, X., ... y Huang, Z. (2020). En oques compu acionales en es udios p eclínicos sob e descub imien o y desa ollo de á macos. F on e as en química , 8 , 726. [13] Jiménez, C. S. H., & Albo, M. V. (2021). Pensamien o compu acional como una habilidad gené ica: una e isión sis emá ica. Ciencia La ina Re is a Cien í ica Mul idisciplina , 5(1), 1055-1078. [14] Cali, D. I. Q., & Peña iel, J. E. R. (2022). Es udio compu acional del compo amien o de dos cue pos con ca gas iguales. Al aPublicaciones, 4(3.2), 6-20. [15] Bu ley, S. K., Be man, H. M., Kleyweg , G. J., Ma kley, J. L., Nakamu a, H., & Velanka , S. (2017). P o ein Da a Bank (PDB): The Single Global Mac omolecula S uc u e A chi e. Me hods in molecula biology (Cli on, N.J.), 1607, 627–641. [16] Be man, H., Hen ick, K., Nakamu a, H., & Ma kley, J. L. (2007). The wo ldwide P o ein Da a Bank (wwPDB): ensu ing a single, uni o m a chi e o PDB da a. Nucleic acids esea ch, 35(Da abase issue), D301–D303. [17] Kim S. (2016). Ge ing he mos ou o PubChem o i ual sc eening. Expe opinion on d ug disco e y, 11(9), 843–855. [18] Kim, S., Chen, J., Cheng, T., Ginduly e, A., He, J., He, S., Li, Q., Shoemake , B. A., Thiessen, P. A., Yu, B., Zasla sky, L., Zhang, J., & Bol on, E. E. (2021). PubChem in 2021: new da a con en and imp o ed web in e aces. Nucleic acids esea ch, 49(D1), D1388–D1395. [19] O'Boyle, N. M., Banck, M., James, C. A., Mo ley, C., Vande mee sch, T., & Hu chison, G. R. (2011). Open Babel: An open chemical oolbox. Jou nal o chemin o ma ics, 3, 1-14. [20] Bi encou -Fe ei a, G., & de Aze edo, W. F. (2019). Docking wi h swissdock. Docking sc eens o d ug disco e y, 189-202. [21] Pa il, N. S., & Rohane, S. H. (2021). O ganiza ion o Swiss Dock: in s udy o compu a ional and molecula docking s udy.