Posted on Authorea 9 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160491622.24853329/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ob cieaantmcbcei hr tsgicnl niie Katvt n a enue spositive as used been has and activity SK inhibited study. development significantly this it in where mycobacteria control against of against active derivative active be and in found to confirmed polyphenol mycobacteria were primarily a polyphenols the . was is few which molecule against mutants.It a enzyme the and the studies benefits. with SK recent of validating health institute CD by against some from the further In active compounds to and be curcumin. binds studies of to specifically docking screening the compound found virtual This during was based well. S-014-1049 structural as domains. where enzyme, culture (SBD) this done binding substrate of was and importance library (CD), CORE the (LD), of to LID Inhibition Owing domains: 3 (SK). of Kinase consist and Shikimate kinases (NMP) is target valuable for for pathway selective most are acid that the shikimic drugs essential for tubercular for enzymes, is fatal precursor pathway is seven a well-recognised chorismate; enzyme involved Among of such this enzymes production of One the inhibitors acids. for of responsible acids. amino discovery reactions amino enzymatic the bacterial seven for aromatic of of made of consisting processes been biosynthesis essential have therapeutic other of the efforts development target extensive for in there to years, way efficient recent drugs targets resistance, and novel In antibiotics promising anti-mycobacterial of is of agents. well-identified discovery screening use drug the the Presently, target-based in Currently, to health. interest survival. Due increased public an to functions. been threat essential has severe a and is structure resistance bacterial drug of development were The compounds 6427 Here MTSK. proven target. of and domain drug H37Rv Introduction core ideal against the an active to it binds found making specifically was mammals, It S-014-1049 in mycobacterium. studies. compound absent in vitro where target is in screening validated in and virtual such non-cytotoxic bacteria and one based disease of is structure the growth kinase through of Shikimate the screened pathogenesis for library. for the techniques chemical the in vital promising defined With involved is the a gene control. of It essential against to one an protein able identifying be the been involves to targeting not It have appears then therapeutics. humans discovery future that drug of menace target-based development a programme, the still discovery is drug disease the oldest in the advancement being of regardless (TB), Tuberculosis Abstract 2020 9, November 1 Dasgupta Arnava Pandey Sapna different with affinity their domains and library Chemical Shikimate from Mycobacterial Kinase against molecules active of Identification eta rgRsac Institute Research Drug Central enovo de ,6 5, ytei fesnilaioacids amino essential of synthesis noeo u rvossuis efuda xsigplpeoi PKC- polyphenolic existing an found we studies, previous our of one In 4 1 kaDhamija Ekta , h ilgcladpamclgclpoete aeplpeosavlal druggable valuable a polyphenols make properties pharmacological and biological The 1 aihna Ramachandran Ravishankar , 7 hrfr,cmon -1-09cudb rmsn rgcniaefrfurther for candidate drug promising a be could S-014-1049 compound Therefore, .tuberculosis M. 1 ajyKumar Sanjay , Mtb ( Mtb ic aml onthv hkmt aha nye necessary enzymes pathway Shikimate have not do mammals since ). . 1 TKblnst h aiyo uloiemonophosphate nucleoside of family the to belongs MTSK Kihbtr ffrptnilfrdvlpeto e anti- new of development for potential offer inhibitors SK 1 1 1 rgaYadav Pragya , n IHR SRIVASTAVA KISHORE and , 1 aiouaNarender Tadigopula , Mtb hc lohv many have also which δ niio,Rottlerin inhibitor, 1 1 , ,3 2, Posted on Authorea 9 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160491622.24853329/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. n otei a sda oiiecnrlfrteasy ucm ftersl showed result the of assay, Outcome assay. ADP-Glo the using for concentrations control varying positive at a S-014-1049 as used against was checked rottlerin was and MTSK of activity with Kinase as well as SKagainstS-014-1049 CD of of activity and residues 1C). several Kinase LEU119 (Figure with PRO118, LD bond of hydrogen are (ARG117) strong at region residue via ligand binding interacts one 3.84 also with compound in with ARG117 interaction this come F3). interaction hydrophobic that which conclude and form pi-anion residues F2 residues form result (F1, other these ASP34 atoms PHE49, binding The atom. fluorine 3.2 interaction. The fluorine with at unfavourable LYS15 with bond ligand an done enzyme. hydrogen the bond compound. was the make the of hydrogen SK also of of (F3-N) forms with SER16 region domains atom S-014-1049 and binding different fluorine of THR17 in to with GLY14, study ARG117 bond compound and Complex hydrogen THR17, this cells. SER16, forms of LYS15, vero GLY14, binding the residues specific to showed tool the non-toxic bioinformatics check using be SK to to index and found selective S-014-1049 was with between S-014-1049 complex Compounds of 16. study than interaction is more Specific S-014-1049 of that 2). index deciphered (Table cytotoxicity selective This candidates most their with drug MABA. for The evaluated appropriate using cells 1B). were concentrations the compounds present (Figure The compound to are SK varying hit. which non-cytotoxic with at each Alanine, affinity with cells and binding bond Vero/THP1 hydrogen Arginine active in form Aspartate, have and Isoleucine, also region active Serine, hits in Glycine, four include the residues all interacted that showed also studies 25 these, at Of inhibition SK. by by against analyzed vitro analyzed hits were in were 134 energy generated energy compounds binding binding 6427 good good of with screening compounds virtual based 44 structure study, our In using and virtual amplified based is Structure DNA plasmid whole the Results where PCR earlier. variant described directions. as a opposite mutants, using phospho-null in created running were primers SK overlapping of mutants of The terms (SDM) in mutagenesis luminometer directed by according Site measured MGIT and was study. MABA kinase using previous done the our was by to screening (RLU).Anti-mycobacterial substrate previous unit of the luminescence relative phosphorylation mentioned reaction, as overall assay 37 of kinase at hour ADP-Glo an through for compounds assessed was MTSK study. of activity and enzymatic H37Rv The screening of anti-mycobacterial 18 DNA (Qiagen). and column at assay genomic Ni-NTA IPTG Enzyme through mM using purified 1.0 were amplified with proteins expressed induced PCR The were was cultures (MSSK), into 3031) 3031 introduced MSMEG and vector (Rv2539; expression gene the M.smegmatis of ORF recombinantSK of expression and Cloning methods and Materials 7 o niiinasy TKwsicbtda ieetcnetain (12.5 concentrations different at incubated was MTSK assay, inhibition For td against study n hnlgtdit Avco.Frhr h eewssbcoe in sub-cloned was gene the Further, vector. TA into ligated then and μ /l(al )adfu eeatv gis 3R t[?]3.12 at H37Rv against active were four and 1) (table g/ml ,8 7, Mtb sn rtcldsrbdearlier described protocol using ° ,sbeunl olwdb diino 2 of addition by followed subsequently C, nvitro in E.coli nvitro in cenn fatv compounds active of screening 8 L1(E)Frepeso fR23 MS)adMSMEG and (MTSK) Rv2539 of expression (DE3).For BL21 tde Fgr A.MB n GTwr sdfrthe for used were MGIT and MABA 1A). (Figure studies 2 pET28a . nvitro in 8 S lsi a sda epaet create to template as used was plasmid :SK fal4 opud ee exhibiting seven compounds 44 all Of ° ,ad08MIT t22 at IPTG 0.8mM and C, > tde.O hs,4 opud with compounds 44 these, Of studies. μ .Smlry eryrsde uhas such residues nearby Similarly, A. ˚ 4 /lsiii cd fe completion After acid. shikimic g/ml itne rmtesuyw can we study the From distance. A ˚ μ /l(al ) h docking The 2). (table g/ml μ pET28a /lt 50 to g/ml itnewihis which distance A ˚ > 0 niiinat inhibition 50% ° respectively. C I vitrogen) (In μ /l of g/ml) > 0are 10 > 55% Posted on Authorea 9 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160491622.24853329/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. rti.Cmaaieaayi ftekns ciiyo uatpoen a oewt epc opurified to respect with done was of proteins activity mutant the of for activity shows domain result kinase essential The the 2 most SK. using of the recombinant performed analysis check was Comparative to Assay assessed assay. protein. was kinase possibly ADP-Glo proteins which using mutant compound SK the the of by proteins activity mutant inhibited Kinase of are activity and kinase CD of conserved SK. Analysis of a CD MS have to and strains binds H37Ra three thatS-014-1049 H37Rv, the suggests 3.12 against all of concentration calculated and Since, a H37Rv ofS-014-1049was 2B). at of MIC inhibition SK showed MABA strains of Using three domains the 2A). analyse (Figure to strains used was software alignment M.smegmatis strains sequence mycobacterial pairwise different Omega of Clustal SK S-014-1049 F). of of 1E, concentration (Figure analysis MIC concentrations 1 domain 2X all Comparative at at at observed rottlerin inhibition was and significant Ra-wt showed S-014-1049 of rottlerin inhibition with whereas Maximum treated blotting. and western taken using was Ra-wt of 4 concentrations culture different phase at Log Ra-wt on S-014-1049 of Effect 50 irr a civd u fwih4 ishdtehgetdcigsoeadwr ute vlae in evaluated further were in-house and the score of docking screening highest virtual the based had structure hits SK, 44 for which inhibitor of specific vitro Out the achieved. find was to between library study LD current and the SBD In CD, namely in SK conserved in of is homolog which domains its three the of all the intervention. absence in among therapeutic present to for are Due SK target of microorganism. promising Orthologs enzyme of a pool fifth viability as large The in serve pathway. a extensively can role shikimate an which of fundamental it late, Against protein plays mammals, Of the clinically SK identified. is approach. pathway, drugs to be screening the of drug resistance to development of structure-based the need increasing for using targets target the screened bacterial drug be evaluated to new can due library the compound been which of for has anti-bacterials tuberculosis. decade of better last prevalence for in of resulting of problem antibiotics decrease useful health available the public in serious influences which Most CD intact of case in Discussion activity occurs enzyme protein, 2E). of the (Figure inhibition activity with Significant enzyme compound control. the a as of used binding was SBD MTSK in type observed (CD mutants wild was 3 and the assay of kinase activity ADP-Glo kinase the of SK role study mutant vital Comparative against its depicting S-014-1049 CD, of of profiling mutation Inhibitory upon significantly SBD (SBD decreases whereas mutated enzyme activity was (LD the SBD enzyme manipulated where of in was protein activity LD the the of where that activity protein kinase suggest the in observed in was observed effect was no decrease 50% around while, nTP1hmnmcohgsadteol opudwihwsfudt enntxcwsS-014-1049 was non-toxic be checked to also found was was compounds which four compound the of only Cytoxicity the S-014- and where compound. assay macrophages most-inhibitory kinase human ADP-Glo the through THP-1 be assessed was in to SK against found compounds was 4 0149 these of activity Specific . μ μ /lfra orwt osatsaig fe ramn yae ftecluewr rprdadanalysed and prepared were culture the of lysates treatment After shaking. constant with hour an for g/ml /lcnetain(iue1D) (Figure concentration g/ml td against study M) twsosre htC a h nydmi hc s0%cnevdi ohthe both in conserved is100% which domain only the was CD that observed was It (MS). Mtb 34 Mtb S,LD :SK, sn AAadMI.Tersl niae opud ob cieagainst active be to compounds 4 indicated result The MGIT. and MABA using n nM hra B n Daesemi-conserved. are LD and SBD whereas MS in and 117 34 S n TKcmae oCD to compared MTSK and :SK S osnthv n ffc nezmtcatvt Fgr 2D). (Figure activity enzymatic on effect any have not does :SK > 0 eraei ciiyo rti hr Dwsmttd(CD mutated was CD where protein of activity in decrease 90% Mtb ope atra iue2 hw arieaineto proteins of alignment pairwise shows 2A Figure bacteria. complex 3 μ /l 12.5 g/ml, 15 μ /lo hkmcai n 0 go purified of ng 600 and acid shikimic of g/ml S,SBD :SK, 9 hs hr sa retncsiyt look to necessity urgent an is there Thus, 15 μ S.Hne tcnb nerdta the that inferred be can it Hence, :SK. /lad25 and g/ml Mtb 34 n S Di h nydomain only the is CD MS. and S,adLD and :SK, μ /lrsetvl (Figure respectively g/ml 34 S) vrl findings Overall :SK). μ 117 117 /l 2 g/ml, S)o Kusing SK of :SK) S) Moreover, :SK). μ /land g/ml 15 . :SK) The Mtb in Posted on Authorea 9 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160491622.24853329/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 1 aihT tkrN.Tecmo rmtcaioai isnhssptwyi seta nMycobac- in essential is pathway biosynthesis acid amino aromatic common The NG. Stoker T, Parish [1] SK, purified for References is bar colour (E). pink assay where kinase SK, purified 50 recombinant at the SBD for (CD to is CD CD comparison green of of in mutant mutant protein mutant is is purified 2 1 of lane (SBD lane Overexpression uninduced, (b) SBD is (B) chromatography of 1 Ni-NTA MABA Lane through using protein concentration. mutant IPTG mycobacteria mM of 1.0 using strains (CD BL21(DE3) three in is (a) against AU protein where S-014-1049 drug densitometry of by no analysed is profile was 1 Blot Lane 2. (E) control. rottlerin Figure 2 a of (0. concentration as The MIC MIC taken MIC. of of was 2X 2X 2X unit(F) 65 1X, and 1X, arbitrary hsp 0.5X, different 0.5X, 1X, where are three are 0.5X, treatment 7 at 4 at drug 6, assay 3, Ra-wt of kinase 2, on hour lanes Glo rottlerin 1 green and ADP with after and using represented observed S-014-1049 SK is was of bond) purified represent types effect action (hydrogen against different structure Inhibitory bond by compounds favorable color (D) interaction the most its concentrations magenta of Where cells,and and SK. profile the of Inhibitory SK, to residue (C) non-cytotoxic is specific be with structure to bonds like found of was ribbon S-014-1049 colour (B). Cyan compounds compound. in individual H37Rv of against active found were 1 Figure (to by is and legends manuscript (DST) Figure this WOS-A to through allotted acceptance). funded number after been communication included has CSIR-CDRI work be The The MLP0120. support. grant CSIR-CDRI the the for CSIR-CDRI Director, thank We AMR. found with were Acknowledgement cope polyphenols to the future of in few candidate molecule. as studies drug druggable recent well valuable the a as polyphenols of make atom some properties against In fluorine active curcumin. with be of derivative to bond and 3.84 hydrogen polyphenol with makes a interaction is also pi-anion complex forms ARG117 the ASP34 both F3). ring. of Hence, atom and benzene study fluorine with F2 with Docking interaction bond (F1, CD. pi-cation hydrogen atoms the forms CD to fluorine of 3.2 compound with residue at defining LYS15 this ligand that in ADP-Glo of of showed three using (F3-N) also binding proteins the S-014-1049 mutant specific among and the showed SK important against the between result checked most Assessing also the is was domain. CD and each S-014-1049 in that assay compound residue of demonstrated important Activity mutants at activity. three mutants the creating the by of analysed activity were kinase SK of domains rottlerin 3 using The 2X) and 1E). 1X (Figure (0.5X, assay MIC blot of control. concentrations positive different a at as H37Ra against checked was compound study same the group susceptible our drug from and study previous In 2). (Table 15 μ S) ae3i uato B (SBD SBD of mutant is 3 lane :SK), /lcnetainaantw Kadmtn K t60gpoencnetainuigADP-Glo using concentration protein 600ng at SKs mutant and SK wt against concentration g/ml ita cenn okflwo nhuelbaycmons()3 ersnaino is(which hits 4 of representation 3D (A) compounds library house in of flow work screening Virtual : oprtv oanaayi fS of SK of analysis domain Comparative nvitro in 34 10 S)adln smtn fL (LD LD of mutant is 3 lane and :SK) 34 hscmon hw euto ncuo uiemdlifce with infected model murine of cfu in reduction shown compound this K e sfrCD for is red SK, : Mtb n okn tde niaebnigo -1-09t Do KCmon S-014-1049 Compound SK of CD to S-014-1049 of binding indicate studies docking and > Mtb 0 niiino xrsinwsosre taltetrecnetain nwestern in concentrations three the all at observed was expression of inhibition 50% n eryrsde uha L1,TR7adSR6as omhdoe bond hydrogen form also SER16 and THR17 GLY14, as such residues nearby and A ˚ n r nw ohv ayhat eet.Tebooia n pharmacological and biological The benefits. health many have to known are and tan n losonsnritceetwt rtln rg ftbruoi.In tuberculosis. of drugs line first with effect synergistic shown also and strains 15 nvitro in Kadylo sfrLD for is yellow and SK : 34 10 S)adln smtn fL (LD LD of mutant is 4 lane and :SK) hscmon a led on cieaantdu resistant drug against active found already was compound this tde)aditrcinwt Kpoenaogwt Dview 2D with along protein SK with interaction and studies) μ g/ μ Mtb )o h opud(-1-09 epciey ae 5, Lanes respectively. (S-014-1049) compound the of l) 4 117 n Syuigbonomtcto A Inhibition (A) tool bioinformatic using MSby and S)rsetvl C iaeasyo h mutant the of assay Kinase (C) respectively :SK) ,5 4, hrfr,ti opudcnb promising a be can compound this Therefore, . 117 KD.Eeto opudS-014-1049 compound of Effect SK(D). : 117 15 S) uicto fthe of Purification :SK). itne(iue1C). (Figure distance A ˚ S) ae2i mutant is 2 lane :SK), Mtb. ffiayo this of Efficacy Posted on Authorea 9 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160491622.24853329/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. their-affinity-with-different-domains of-active-molecules-against-mycobacterial-shikimate-kinase-from-chemical-library-and- 2.pdf Table file Hosted their-affinity-with-different-domains of-active-molecules-against-mycobacterial-shikimate-kinase-from-chemical-library-and- 1.pdf Table novel of Future evaluation file Biological Hosted al. tuberculosis. et Mycobacterium SK, Puttrevu drug-resistant P, 2017. Gupta against Microbiol VK, active Singh Innovare P, derivative pathway. Karaulia kinase curcumin-pyrazole-mannich P, shikimate Yadav into AK, insight Singh critical [10] and A P. Biochemical Patyal Appl SK, 2015;7:25–7. Thokchom Sciences al. SS, inhibitors. Academic Mehta et phosphatase S, R, tyrosine Mehta mycobacteria: [9] Ramachandran as nonpathogenic azoles N, and butenyl Rai 2015;99:7539–48. pathogenic methyl-/phenyl Biotechnol NP, cyclopropyl from Microbiol Pathak phenyl phosphatases C- PK, of tyrosine kinase Singh indication of Protein S, characterizations KK. Pandey Srivastava functional A, R, Chatterjee Ramachandran [8] S, 2016;478:721–6. Kumar Commun Res S, Biophys Jaiswal Biochem Planta A, opportunities. Chatterjee and Δ S, weaknesses Strengths, Pandey - [7] resistance bacterial actions https://doi.org/10.1055/s-2008-1074518. for anti-diabetic 2008;74(6):594-602 Phytochemicals the Med., understanding S. in 2013;24(11):1777-1789 Gibbons advances Biochem 2011;87:66–80. [6] Recent Nutr Sci ER. J Biol Gilbert n.d. Phys D, flavonoids B Liu dietary Ser A, of Acad Babu Jpn P, Proc Velayutham assay sensing. [5] Chem polyphenol ESI-LC-MS-based Anal tea an Green inhibition. of H. and Tachibana Development activity [4] Calder´on AI. kinase DC, shikimate tuberculosis Goodwin mycobacterium Y, 2015;87:2129–36. of Wang evaluation G, functional kinetic of Gill for form J, soluble Simithy in tubercu- [3] overexpression Mycobacterium 2001;22:430-435 and from Purif Cloning enzymes Expr DS. synthase Protein Santos 3-phosphate losis. 5-enolpyruvylshikimate LA, and Basso kinase CA, shikimate Pinto JS, Oliveira [2] 2002;148:3069–77. Microbiology tuberculosis. terium niio,Rtlrnihbt rwhadsria fmcbcei xlsvl hog hkmt kinase. Shikimate through exclusively mycobacteria of survival and growth inhibits Rottlerin inhibitor, vial at available at available https://authorea.com/users/374103/articles/491726-identification- https://authorea.com/users/374103/articles/491726-identification- 5 Posted on Authorea 9 Nov 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160491622.24853329/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 6