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
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