Supplementary Note 1. A human fatty acid synthase inhibitor binds -ketoacyl reductase in the ketosubstrate

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1 uppleentary Note huan fatty acd synthase nhbtor bnds -ketoacyl reductase n the ketosubstrate ste Mary nn Hardwcke,4, lan R. Rendna 2,4, hawn P. Wllas 3,4, Mchael L. Moore, Lpng Wang 3, ule. rueger, Raona N. Plant, Rachel D. Totorts 2, Guofeng Zhang 2, acques rand 2, Wlla. urkhart 3, rstn. rown 2 & Cyntha. Parrsh * Oncology R&D, Glaxothlne, Collegelle, Pennsylana, U. 2 Platfor Technology and cence, Glaxothlne, Collegelle, Pennsylana, U. 3 Platfor Technology and cence, Glaxothlne, Research Trangle Park, North Carolna, U. 4 These authors contrbuted equally to ths work. *e-al: cyntha.a.parrsh@gsk.co ssay of partal and oerall hf acttes wth coenzye substrates. The actty and nhbton of the oerall F reacton and 3 of the partal reactons were easured contnuously at abent teperature n 96-well plates usng the decrease n 340 n absorbance due to NDPH oxdaton. C 50 alues were deterned n the presence of 40 M c-co, 40 M M-Co and 60 M NDPH for the oerall reacton at 5 nm hf, 40 M acetoacetyl-co and 60 M NDPH for R at 5 nm hf, 40 M - hydroxybutyryl-co and 60 M NDPH for DH at 00 nm hf, and 40 M crotonyl- Co and 60 M NDPH for ER at 00 nm hf as descrbed preously. The partal acttes usng acyl-co substrates requre the actty of the MT doan to load these substrates onto the CP, and thus deterne nhbton of both MT plus R, MT plus DH, or MT plus ER. The total reacton olue was 300 L of 50 M sodu phosphate, ph 7.0 contanng 0.04% Tween-20, and 0.05 to 000 nm G n 3-fold ncreents for the C 50 deternatons. Data were conerted to % actty by coparson to a no nhbtor control and ftted to a standard 4 paraeter nhbton odel. For the MO studes of R, acetoacetyl-co was ared fro.0 to 50 M at fxed NDPH (30 and 20 M), NDPH was ared fro 2 to 250 M at fxed acetoacetyl- Co (20 and 300 M), and G was ared fro 0 and.6 to 00 nm n 2-fold ncreents at 5 nm hf. To correct for non-enzyatc oxdaton of NDPH, controls were conducted n the absence of hf. n Enson Multlabel Reader (PerknEler) was used to ontor absorbance changes. No sgnfcant nhbton was obsered for ether the MT plus DH or MT plus ER actty at 000 nm G ssay of and TE partal hf acttes. The cobned actty of MT plus was deterned spectrophotoetrcally usng a Molecular Deces plate reader at 283 n to detect the foraton of the tracetc acd lactone condensaton product catalyzed by 200 nm hf fro 40 M c-co and 40 M M-Co n the absence of NDPH 2. The total reacton olue n 384-well Grener U tar crotter plates was 00 L of 50 M sodu phosphate, ph 7.0 contanng 0.04% Tween-20, and 0, and 0 M G (% DMO fnal) n duplcate. Data were conerted to % actty by Nature Checal ology: do:0.038/nchebo.603

2 coparson to a no nhbtor control after subtracton of a no hf control. The TE actty was deterned n a Molecular Deces nalyst plate reader by easurng the hydrolyss of the fluorescent substrate 4-ethylubellferone (4-MUH; ga-ldrch) through exctaton at 360 n and esson at 460 n usng a 425 n dchroc rror by a odfcaton of the ethod descrbed n Rchardson and th 3. The total reacton olue n 384-well Grener black crotter plates was 50 L of 50 M sodu phosphate, ph 7.0 contanng 0.04% Tween-20, 50 M 4-MUH, 200 nm hf and 0, and 0 M G (% DMO fnal) n duplcate. Data were conerted to % actty by coparson to a no nhbtor control after subtracton of a no hf control. No sgnfcant nhbton of ether MT plus or TE was obsered at 000 or 0,000 nm G ssay of R actty wth trans--decalone. The nhbton echanss for nhbtors of the R doan were deterned by contnuously ontorng the oxdaton of NDPH by decrease n absorbance at 340 n n the presence of trans--decalone (ga- ldrch). The concentraton of one of these two substrates was ared, and the second substrate was fxed at ether saturatng or sub-saturatng concentratons n the presence of ared nhbtor concentratons. Reactons were carred out at abent teperature n 96- well ½ area Cornng 3884 crotter plates n a fnal olue of 00 or 50 L contanng 50 M sodu phosphate at ph 7.0, 0.04% Tween-20, 3 or 4 nm huan full length F typcally, and arable concentratons of NDPH (typcally fro 6 to 00 M), trans-- decalone (typcally 0.5 to 8 M fro a M stock and seral dlutons n DMO), NDP + (typcally fro 2.5 to 400 M), G (typcally fro to 80 nm fro a 0 M stock and seral dlutons n DMO), and G83749 (typcally fro 52 to 833 nm fro a 0 M stock and seral dlutons n DMO) wth -2% DMO fnal. For the MO studes wth the hf tr-doan crystallographc proten, the enzye concentraton was 0 nm, trans--decalone was ared fro 0.8 to 2.8 M, and G was ared fro 50 to 800 nm. n the dual substrate ttraton experent descrbed n uppleentary Fgure 2a, hf was 5 nm, trans-- decalone was ared fro to 0 M and NDPH was ared fro 2.5 to 200 M. For the deternaton of utually excluse bndng of NDP + and G by Yonetan- Theorell analyss shown n Fgure d, hf was 4 nm, NDP + was ared fro 0 and 2.5 to 400 M, and G was ared fro 0 and 5 to 80 nm. Controls were carred out n the absence of F to correct for non-enzyatc oxdaton of NDPH. bsorbance changes were ontored usng an Enson Multlabel Reader (PerknEler). Data nalyss. alues for the knetc paraeters and ther standard errors were obtaned by fttng data to the approprate equatons usng the nonlnear regresson functon of GraFt (Erthacus oftware). The ntal rate data for and M deternatons were ftted to eqs and 2. Data fro echans of nhbton studes were ftted to eqs 3-6 and for the Yonetan-Theorell analyss to eqs 7-8. The best odel was deterned fro the lowest ch squared alue and lowest standard errors n the ftted paraeters. 2 Nature Checal ology: do:0.038/nchebo.603

3 3 () Mchaels-Menten a b b a (2) two substrate sequental echans (3) copette nhbton s (4) noncopette nhbton; separate s (5) noncopette nhbton wth s = (6) uncopette nhbton j j beta (7) nonexcluse nhbton j (8) utually excluse nhbton n equatons to 8, s the ntal elocty, s the u elocty,,, and are the ared substrate concentratons, s the nhbtor concentraton, a, b and are Mchaels constants, a s the dssocaton constant for wth alpha = a / a, and, j, s and are nhbton constants where s s the dssocaton constant for the E- coplex and s the dssocaton constant for fro the E-- coplex when s saturatng. n equatons 7 and 8, or equatons 3-6 when the fxed substrate was not saturatng (uppleentary Table, footnote b), and are the nhbtor concentratons for the two nhbtors, and j are the apparent nhbton constants (C 50 alues) under the experental condtons and beta s the nteracton coeffcent, typcally < for synergstc bndng, for ndependent bndng and > for antagonstc bndng. The best ft for the data shown n Fgure b was to eq 6 for uncopette nhbton ersus NDPH wth = /n, NDPH app M = 8.7 Nature Checal ology: do:0.038/nchebo.603

4 0.60 M, and G app = 7.0 nm, whle the data for Fgure c ft best to eq 3 for copette nhbton ersus trans--decalone wth = /n, decalone M = M and G = nm. Double nhbton data for Fgure d yelded a seres of parallel lnes n a plot of /ntal rate as a functon of NDP + concentraton and ft best to eq 8 for utually excluse nhbton between NDP + and G wth = /n, NDP + app = M and G app j = 33.6 nm. Reersblty Experents for G uppleentary Fgure 2a shows an exaple of the te courses for NDPH oxdaton of 2 nm hf R actty n the presence of 48 nm G (squares) and 4 M trans--decalone copared to a.2% DMO control (trangles). ssays were set up as descrbed n uppleentary Fgure 2b for the no pre-ncubaton controls. The te courses are lnear for at least 20 n ndcatng no te-dependent nhbton wth a one nute xng te. uppleentary Fgure 2b: n duplcate wells of a Cornng ½ area 96-well crotter plate 0 L of concentrated G dlutons (0 fnal) n 0% DMO assay buffer or 0% DMO assay buffer n control wells was added to 50 L of 4 nm hf contanng 200 M NDPH n assay buffer (50 M sodu phosphate, ph 7.0 wth 0.04% Tween-20). fter pre-ncubaton perods of h, 2 h, 4.7 h and 7 h, 40 L of 0 M trans--decalone was added and the absorbance at 340 nm was ontored contnuously eery 8 sec for 60 n n a Perkn Eler Enson crotter plate reader. The no pre-ncubaton controls were prepared by addng the decalone to the wells pror to addng the F/NDPH xture. The fnal concentratons n the 00 L assay were 2 nm hf, 0 nm for the control wells (aerage of 8) or to 300 nm G n 2.5-fold ncreents,.2% DMO, 4 M trans--decalone and 00 M NDPH. C 50 alues were calculated usng a standard 4-paraeter odel and plotted as % DMO Control ctty after subtracton of an aerage no decalone background rate (8 wells) ersus the G concentraton on a logarthc scale. s shown n the legend for uppleentary Fgure 2b, the C 50 alues do not change sgnfcantly wth ncreasng pre-ncubaton te and range fro 3 to 6 nm. uppleentary Fgure 2c: brefly, 00 nm hf was ncubated wth 400 nm nhbtor (or % DMO n the control) and saturatng NDPH (00 M) for h and 4 h at roo teperature n a total olue of 00 L of 50 M sodu phosphate, ph 7.0 contanng 0.04% Tween-20 to ensure foraton of the nhbted NDPH coplex. fter the pre-ncubaton te perod,.5 L alquots of treated and untreated saples were added to 8 replcates of 50 L (0-fold dluton) of assay xture contanng 2 M trans--decalone and 00 M NDPH n 50 M sodu phosphate, ph 7.0 contanng 0.04% Tween-20. The absorbance at 340 n was ontored contnuously eery 8 sec for up to h usng a Perkn Eler Enson crotter plate reader. ntal rates were calculated between 0 and 20 n and aeraged (n = 6 to 8). For the h pre-ncubaton 77% of the actty was recoered fro G treated saples copared to the % DMO controls, whle for the 4 h prencubaton 84% actty was recoered. Due to carry oer of 4 nm G after dluton, the theoretcal u recoery was 89% based on the calculated C 50 fro copetton wth trans--decalone at 2 M usng a of 0 nm and M of 5.9 M (see below). Thus, experental recoeres were close to expected. 4 Nature Checal ology: do:0.038/nchebo.603

5 topped-flow Deternaton of G Mcroscopc Rate Constants Experents were conducted by rapdly xng equal olues of 40 nm hf n 50 M sodu phosphate buffer, ph 7.0 wth 80 M NDPH, 8 M trans--decalone and 2 tes the arable G ndcated n uppleentary Fgure 3a usng an ppled Photophyscs X20 stopped-flow spectroeter. Obsered rate constants at each nhbtor concentraton (n = 2 to 4) except zero (lnear ft) were calculated fro fts of the cured te courses (cured at less than 30 sec) to the equaton below descrbng product foraton n the presence of slow bndng nhbtors 4 : P = s t + ( 0 s )( e -kt )/k where P represents product (absorbance at 340 n), s and 0 represent the fnal steady state and ntal eloctes, respectely, and k s the apparent frst order rate constant. The obsered rate constants (k obs ) were plotted ersus concentraton of nhbtor (uppleentary Fg. 3b) and ftted to a lnear equaton to ge k on fro the slope after adjustng for substrate copetton (k on = slope( + decalone/ M ) and k off drectly fro the ntercept. The decalone M ( M) used n the calculaton was an aerage of 29 deternatons. REFERENCE. azquez, M.. et al. Dscoery of G83749, an nhbtor of huan fatty acd synthase targetng the -ketoacyl reductase reacton. FE. 275, (2008). 2. Rendna,.R. & Cheng, D. Characterzaton of the nactaton of rat fatty acd synthase by C75: nhbton of partal reactons and protecton by substrates. oche.. 388, (2005). 3. Rchardson, R.D. & th,.w. Noel antagonsts of the thoesterase doan of huan fatty acd synthase. Mol. Cancer Ther. 6, (2007). 4. Morrson,. F. & tone,.r. pproaches to the study and analyss of the nhbton of enzyes by slow- and tght-bndng nhbtors. Coents Mol. Cell ophys. 2, (985). 5 Nature Checal ology: do:0.038/nchebo.603