St r uctur al sim i l ar i t i es between som e com m on f l uor ophor es used in biology and m ar keted dr ugs, endogenous m etabol i tes, and natur al pr oducts Steve O’Hagan 1,2, Douglas B. Kel l 1,2,3,4*

1 School of Chemistr y, 2Manchester Insti tute of Biotechnology, The Univer si ty of Manchester , 131 Pr incess St, Manchester M1 7DN, UK

3 (and pr esent addr ess): Depar tment of Biochemistr y, Insti tute of Integr ative Biology, Biosciences Bui lding, Univer si ty of Liver pool, Cr own Str eet, Liver pool L69 7ZB, UK.

4 Novo Nor disk Foundation Centr e for Biosustainabi l i ty, Technical Univer si ty of Denmar k, Bui lding 220, Kemitor vet, 2800 Kgs. Lyngby, Denmar k

* Cor r espondence: Douglas Kel l , Emai l : dbk@liv.ac.uk; Tel.: +44-151-795-7772; @dbkell http://dbkgr oup.or g/

ABSTRACT

Background: It is kn ow n th a t a t least som e flu or oph or es can act a s ‘su r r oga te’ su bstr a tes for solu te ca r r ier s (SLCs) in volved in ph a r m aceu tica l d r u g u p take, an d th is p r om iscu ity is taken to r eflect a t least a cer ta in str u ctu r a l sim ila r ity. As pa r t of a com pr eh en sive stu dy seekin g th e ‘n a tu r a l’ su bstr a tes of ‘or ph an ’ tr an spor ter s th a t a lso ser ve to take u p ph a r m aceu tica l d r u gs in to cells, w e h ave n oted th a t m an y d r u gs bea r str u ctu r a l sim ila r ities to n a tu r a l p r odu cts. A cu r sor y in spection of com m on flu or oph or es in d ica tes th a t th ey too a r e su r p r isin gly ‘d r u g-like’, an d th ey a lso en ter a t least som e cells. Som e a r e a lso kn ow n to be su bstr a tes of efflu x tr an spor ter s. Con sequ en tly, w e sou gh t to a ssess th e str u ctu r a l sim ila r ity of com m on flu or oph or es to m ar keted d r u gs, en dogen ou s m am m alian m etabolites, an d n a tu r a l p r odu cts. We u sed a set of som e 150 flu or oph or es. Results: Th e gr ea t m a jor ity of flu or oph or es tested exh ib ited sign ifican t sim ila r ity (Tan im oto sim ila r ity > 0.75) to a t least on e d r u g a s ju dged via descr ip tor p r oper ties (especia lly th eir a r om aticity, for iden tifiab le r eason s th a t w e exp la in ), by m olecu la r fin ger p r in ts, by visu a l in spection , an d via th e “qu an tita tive estim a te of d r u g liken ess” tech n iqu e. It is con clu ded th a t th is set of flu or oph or es does over lap a sign ifican t par t of both d r u g space an d n a tu r a l p r odu cts space. Con sequ en tly, flu or oph or es do in deed offer a m u ch w ider oppor tu n ity th an h ad possib ly been r ea lised to be u sed a s su r r oga te u p take m olecu les in th e com petitive or tr an s-stim u la tion a ssay of m em br an e tr an spor ter activities.

KEYWORDS: dr u gs – n a tu r a l p r odu cts – flu or oph or es – sim ila r ity – ch em in for m a tics – Tan im oto d istan ce

ABBREVIATIONS

PCA, Pr in cipa l Com pon en ts An a lysis; QED, Qu an tita tive Estim a te of Dr u g-liken ess; QSAR, Qu an titta ive Str u ctu r e-Activity Rela tion sh ip ; SLC, solu te ca r r ier ; TS, Tan im oto sim ila r ity; UNPD, Un iver sa l Na tu r a l Pr odu cts Da tabase

INTRODUCTION

Flu or escen ce m eth ods h ave been u sed in b iologica l r esear ch for decades, an d th eir u tility r em a in s u n aba ted (e.g. [1-16]). Ou r specific in ter est h er e is in th e tr an spor ter -m edia ted m ean s by w h ich sm a ll flu or escen t m olecu les en ter livin g cells, an d ou r in ter est h as been stim u la ted by th e r ecogn ition th a t a given pr obe m ay be a su bstr a te for a la r ge va r iety of both in flu x an d efflu x tr an spor ter s [17]. Efflu x tr an spor ter s a r e often fa ir ly p r om iscu ou s, sin ce th eir job is la r gely to r id cells of u n w an ted m olecu les th a t m ay h ave en ter ed , a lth ou gh th ey can an d do h ave oth er , im por tan t ph ysiologica l r oles (e.g. [18-28]), an d

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

th ey a r e capab le of efflu xin g a var iety of flu or escen t pr obes (e.g. [29-36]). How ever , given th a t m ost of th ese p r obes a r e con tem por ar y, syn th etic m olecu les, th e u p take tr an spor ter s for w h ich th ey a r e su bstr a tes m u st h ave evolved in n a tu r e for oth er pu r poses. Th ese pu r poses m ay r eason ably be expected to in clu de th e u p take of en dogen ou s m etabolites in m u lticellu la r or gan ism s [37-40], a s w ell a s exogen ou s n a tu r a l p r odu cts w h ose u p take can en h an ce b iologica l fitn ess (e.g. [41; 42]). Th is explan a tion does seem s to h old w ell for syn th etic, m ar keted ph a r m aceu tica l d r u gs [42].

Con sequ en tly, it seem ed r eason ab le th a t existin g flu or escen t m olecu les, n ot specifica lly design ed for th e pu r pose bu t th a t a r e taken u p by b iologica l cells, m igh t a lso bea r str u ctu r a l sim ila r ities to en dogen ou s su bstr a tes (m etabolites) an d to n a tu r a l p r odu cts, an d poten tia lly a lso to m ar keted d r u gs. If so, th ey m igh t th en ser ve a s su r r oga te tr an spor ter su bstr a tes for th em . In deed , th er e a r e exam ples – so-ca lled flu or escen t fa lse n eu r otr an sm itter s – w h er e su ch flu or escen t an a logu es of n a tu r a l su bstr a tes h ave been design ed p r ecisely for th is pu r pose (e.g. [43; 44]). Th e a im of th e p r esen t w or k w as to a ssess th e exten t to w h ich th is kin d of str u ctu r a l sim ila r ity betw een (i) com m on flu or oph or es u sed in b iology an d (ii) oth er m olecu la r cla sses (en dogen ou s m am m alian m etabolites, m ar keted ph ar m aceu tica l d r u gs, an d kn ow n n a tu r a l p r odu cts) m igh t be tr u e. It is con clu ded th a t in str u ctu r a l ter m s com m on flu or oph or es do in deed over lap d r u g space sign ifican tly, an d w e offer an exp lan a tion based on th e con son an ce betw een a r om aticity, con ju ga ted π-bon ds, an d flu or escen ce.

MATERIALS AND METHODS

Fluor ophor es w er e selected fr om the l i ter atur e and by scanning var ious catalogues of f luor ophor es, and included w ell known cytochemical stains, food dyes, laser dyes and other f luor ophor es, including just a few marketed dr ugs plus f luor escent natur al pr oducts. We chose only those w hose str uctur es w er e known publicly. The f inal set included 150 molecules. Supplementar y Table 1 gives a spr eadsheet of al l the r elevant data that w e now discuss, including the marketed drugs, Recon2 metaboli tes [45] (both given also in r ef 37) and a subset of 2000 natur al pr oducts fr om UNPD (see [42; 46]).

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Figur e 1. Pr incipal components and t-SNE plots of the pr incipal components of the var iance in calculated pr oper ties of the molecules used. A. The f i r st tw o pr incipal components of the var iance in calculated pr oper ties of the four classes f luor ophor es, dr ugs, metaboli tes and natur al pr oducts. Molecules ar e as in Table S1, w ith dr ugs and metaboli tes being those given in [37]. A sampling of 2000 natur al pr oducts fr om our dow nload [42] of UNPD w as used. Descr iptor s w er e z-scor es nor malised and cor r elation f i l ter ed (thr eshold 0.98. B. t-SNE plot of the data in (A), using the same colour coding. C. Plot of the f ir st tw o pr incipal components of the var iance of the f luor ophor es alone. The excitation w avelength is encoded in the colour of the mar ker s. The size of the symbol encodes the molecular w eight, indicating that much of the f i r st PC is due to this (plus any other covar ying pr oper ties).

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Alth ou gh th er e a r e a gr ea t m an y possib le m olecu la r en cod in gs (w h eth er u sin g m olecu la r fin ger pr in ts or vector s of ca lcu la ted pr oper ties), each of w h ich can give a d iffer en t Tan im oto sim ila r ity, for ou r pr esen t pu r pose w e ch ose to u se on ly th e Pa tter n ed en cod in g w ith in RDKit (w w w .r dkit.or g/). We a lso u sed th e RDKit ver sion of QED (h ttps://w w w .r dkit.or g/docs/sou r ce/r dkit.Ch em .QED.h tm l). Wor kflow s w er e w r itten in KNIME as per ou r stan da r d m eth ods [37-40; 42; 47-49]. t-SNE p lots u sed th e fir st 10 PCs (95.3% exp la in ed va r ian ce) a s in pu ts based on 27 RDKit descr ip tor s, an d w er e oth er w ise a s p r eviou sly descr ibed [50].

RESULTS

Figur e 1A gives a Pr incipal Components Analysis (PCA) plot of the distr ibution of the four classes based on a ser ies of descr iptor s in RDKit (w w w .r dki t.org/), w hi le Fig 1B gives a t-SNE [51] plot of the same data. These clear ly show a str ong over lap betw een the r ather l imited set of f luor ophores used and quite signif icant par ts of dr ug space. Fig 1C gives just the f luor ophor es, w ith the nominal exci tation maximum encoded in i ts colour . This suggests that even w ith just ~150 molecules w e have achieved a r easonable cover age of the r elevant ‘f luor ophor e space’, w ith no obvious bias, nor tr end in exci tation w avelengths.

We pr eviously developed the use of r ank order plots for summar ising the r elationships (in terms of Tanimoto simi lar i ties) betw een a candidate molecule or set of molecules and a set of tar gets in a l ibr ar y [37]. Fig 2 show s such a rank or der plot, r anking for each f luor ophor e the most simi lar molecule in the set of endogenous Recon2 [37; 52] metaboli tes, the set of mar keted drugs [37], and a r andom subset of 2000 of some 150,000 molecules taken [42; 49] fr om the Unif ied Natural Pr oducts Database (UNPD) [46]. This again show s ver y clear ly that the major i ty of f luor ophor es chosen do look moder ately simi lar (TS>0.75) to at least one dr ug (and even mor e so to r epr esentatives of the natur al pr oducts database).

Figur e 2. Ran ked or der of Tan im oto sim ila r ity for flu or oph or es vs m ar keted d r u gs (−−−−−−),

flu or oph or es vs Recon 2 m etabolites (−−−−−−), an d flu or oph or es vs a 2000-m em ber sam plin g of UNPD

(−−−−−−). Each flu or oph or e w as en coded u sin g th e RDKit ‘Pa tter n ed’ en cod in g, th en th e Tan im oto sim ila r ity for it ca lcu la ted aga in st each d r u g, m etabolite or n a tu r a l p r odu ct sam ple. Th e h igh est va lu e of TS for each flu or oph or e w as r ecor ded an d th ose va lu es r an ked . Read fr om r igh t to left.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

It is a lso con ven ien t [37] to d isp lay su ch da ta a s a h ea t m ap [53], w h er e a b iclu ster is u sed to clu ster sim ila r str u ctu r es an d th e colou r of th e cell a t th e in ter section en codes th eir Tan im oto sim ila r ity. Figu r e 3 sh ow s su ch h ea tm aps for (A) flu or oph or es vs en dogen ou s (Recon 2 [45]) m etabolites, (B) dr u gs, an d (C) 2000 sam pled n a tu r a l p r odu cts fr om UNPD. Th e da ta r eflect th ose of Fig 2, an d it is aga in clea r th a t for each flu or oph or e th er e is a lm ost a lw ays a d r u g or a n a tu r a l p r odu ct for w h ich th e aver age Tan im oto sim ila r ity is sign ifican tly gr ea ter th an 0.7.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Figur e 3. Heat maps i l lustr ating the Tanimoto simi lar i ties (using the RDKit patter ned encoding) betw een our selected f luor ophor es and (A) Recon2 metaboli tes, (B) Drugs, and (C) a subset of 2000 natur al pr oducts fr om UNPD.

While is r ather arbi tr ar y, to say the least (given how the Tanimoto simi lar i ty var ies w ith the encoding used), as to w hether a par ticular chemical str uctur e is seen by humans as ‘simi lar ’ to another , w e pr ovide some i l lustrations that give a feel ing for the kinds of simi lar i ty that may be obser ved.

Thus (Fig 4A) w e i l lustr ate the dr ugs closest to f luor escein in t-SNE space (as per Figur e 1B), since f luor escein is a ver y common f luorophor e, is also w idely used in ophthalmology (e.g. [54; 55]), and can enter cel ls via a var iety of tr anspor ter s [56] such as monocar boxylate tr anspor ter s (SLC16A1, SLC16A4) [57], SLCO1B1/3B1 [58; 59] and SLC 22A20 [60] (see also Table 1).

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Figur e 4. Observable str uctur al simi lar i ties betw een selected f luor ophor es and dr ugs. The chosen molecules ar e (A) f luorescein, (B) dapoxyl (both f luor ophor es) and (C) ni tisinone (a dr ug). Data ar e annotated and/or zoomed fr om those in Fig 1B.

Fluor escein is simi lar in t-SNE space (Fig 4A) to a var iety of drugs. This simi lar i ty is not at al l r elated to the class of dr ug, how ever , as close ones include balsalazide (an anti-inf lammator y used in inf lammator y bow el disease [61]), bentir omide (a peptide used for assessing pancr eatic function [62]), butenafine (a topical anti fungal [63]), ser tindole (an atypical antipsychotic), and tolvaptan (used in autosomal dominant polycystic kidney disease [64]). Simi lar r emarks may be made of dapoxyl (Fig 4B). Note, of cour se, that the t-SNE plots ar e based on pr oper ty descr iptor s, w hile the Tanimoto distances ar e based on a par ticular form of molecular f ingerpr int, so a pr ior i w e do not necessar i ly expect the closest molecules to be the same in

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

th e tw o cases. In add ition , w e n ote th a t m olecu les w ith d iffer en t sca ffolds m ay be qu ite sim ila r ; in th e ch em in for m a tics liter a tu r e th is is kn ow n as ‘sca ffold h opp in g’ (e.g. [65-70]).

For a dr u g, w e p icked n itisin on e, a d r u g active aga in st h er ed ita r y tyr osin aem ia type I [71] an d a lkap ton u r ia [72; 73], a s it is su r r ou n ded in t-SNE space (Fig 4C) by sever a l tr icyclic flu or oph or es, th a t do in deed sh ar e sim ila r str u ctu r es (Fig 4C).

Bicker ton an d colleagu es [74] in tr odu ced th e con cep t of th e qu an tita tive estim a te of dr u g-liken ess (QED) (bu t see [75]), an d it is of in ter est to see h ow ‘d r u g-like’ ou r fou r cla sses of m olecu le a r e by th eir cr iter ia . Fig 5A sh ow s th e d istr ibu tion of QED dr u g-liken esses for m ar keted d r u gs, for Recon 2 m etabolites, for ou r selected flu or oph or es, an d for a sam ple of 2000 m olecu les fr om UNPD. Ou r flu or oph or es a r e n oticeab ly m or e sim ila r to dr u gs th an a r e en dogen ou s m etabolites, an d r ou gh ly as sim ila r to d r u gs a s a r e n a tu r a l p r odu cts (Fig 5A).

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Figur e 5. Distr ibution of quanti tative estimate of dr ug-l ikeness (QED) values in di f fer ent classes of molecule. A. Cumulative distr ibutions for the four classes. B. Relationship betw een QED and ar omatici ty for th e fou r cla sses a s en coded by th e fr action of C a tom s exh ib itin g sp 3 bon din g. QED va lu es w er e ca lcu la ted u sin g th e RDKit Pyth on code a s descr ibed in Meth ods an d p lotted in (A) u sin g ggp lot2 an d in (B) u sin g Spotfir e. C. Den sity d istr ibu tion of fr action of C a tom s w ith sp 3 bon d in g. D. Histogr am of d istr ibu tion s of n u m ber s of a r om atic r in gs in th e fou r given cla sses.

Given th a t essen tia lly a ll d r u gs a r e sim ila r to a t least on e n a tu r a l p r odu ct [42], th is is en tir ely con sisten t

w ith ou r th esis th a t m ost flu or oph or es do look r a th er like on e or m or e m ar keted d r u gs. On e a spect in w h ich (a ) d r u gs an d flu or oph or es d iffer n oticeab ly fr om (b) m etabolites an d n a tu r a l p r odu cts is th e exten t to w h ich th ey exh ib it a r om aticity, h er e en coded (Fig 5B, on th e abscissa ) via th e fr action of ca r bon a tom s sh ow in g sp 3 h ybr id isa tion (i.e. n on -a r om atic). Th is is sh ow n as a d istr ibu tion in Fig 5C. Th er e is clea r ly a sign ifican t ten den cy for d r u gs to in clu de (p lan a r ) a r om atic r in gs, an d a lth ou gh th is is ch an gin g som ew h a t [76-80] th er e a r e str on g th er m odyn am ic r eason s a s to w h y th is sh ou ld be so (see Discu ssion ). Th e m oda l n u m ber of a r om atic r in gs for both d r u gs an d flu or oph or es is tw o, sign ifican tly gr ea ter th an th a t (zer o) for m etabolites an d for n a tu r a l p r odu cts (Fig 5D). On e r eason for flu or oph or es to exh ib it a r om aticity is sim ple, a s r eason able visib le-w avelen gth flu or escen ce in or gan ic m olecu les r elies str on gly on con ju ga tion (e.g. [81]), to w h ich a r om atic r in gs can con tr ibu te str on gly. Th is a r gu m en t a lon e pr obably accou n ts in la r ge m easu r e for th e d r u g-liken ess of flu or oph or es.

Fin a lly, a ver y r ecen t, p r in cip led , an d effective clu ster in g m eth od [82; 83], r ep r esen tin g th e sta te of th e a r t, is th a t based on th e Un ifor m Man ifold Appr oxim a tion an d Pr ojection (UMAP) a lgor ith m . In a sim ila r vein , an d based on th e sam e descr ip tor s a s u sed in th e t-SNE p lots, w e sh ow th e clu ster in g of ou r fou r cla sses of m olecu le in UMAP space, w h er e m ost clu ster s con ta in in g d r u gs a lso con ta in flu or oph or es. Desp ite bein g based on p r oper ty descr ip tor s, th e UMAP a lgor ith m is clea r ly ver y effective a t clu ster in g m olecu les in to str u ctu r a lly r ela ted classes.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Fig 6. UMAP pr ojection in to tw o d im en sion s of th e fou r cla sses of m olecu les, an n ota ted by th e type of

m olecu la r str u ctu r e in th e va r iou s clu ster s.

DISCUSSION

The basis of the main idea pr esented her e is that the str uctur es of common f luor ophor es ar e suff iciently simi lar to those of many dr ugs as to pr ovide suitable sur r ogates for assessing their uptake via solute car r ier s of the SLC (and indeed their eff lux via ABC) famil ies. Whi le the latter tr anspor ter s ar e w ell known to be r ather pr omiscuous, and to tr anspor t a var iety of f luor ophor es [34; 36; 84-86], considerably less attention has been paid to the for mer . Of cour se some mar keted pharmaceutical dr ugs that ar e tr anspor ted into cel ls are in fact natur ally f luor escent, including molecules such as anthr acycl ines [87-89], mepacr ine (atebr in, quinacr ine) [90], obatoclax [91; 92], tetr acycl ine der ivatives [88; 93] and topotecan [94], The same is tr ue of cer tain vi tamins such as r ibof lavin [95; 96] (that necessar i ly have tr anspor ter s, as cel ls cannot synthesise them), as w ell as cer tain bioactive natur al pr oducts (e.g. [97-99]). As an i l lustr ation, and as a complement to our detai led gene knockout studies [17], Table 1 gives an indication of dyes w hose inter action w ith specif ic tr anspor ters has been demonstrated dir ectly. In some cases, their sur r ogacy as a substrate for a tr anspor ter w ith a know n non-f luor escent substr ate is clear , and as mentioned in the intr oduction they ar e sometimes r efer r ed to as ‘false f luor escent substr ates’. Overal l, w hi le not intended to be r emotely exhaustive, this Table does ser ve to indicate the potential ly w idespr ead activi ty of tr anspor ter s as mediators of f luor ophor e uptake, and indeed a number of such tr anspor ter s ar e know n to be r ather promiscuous.

Table 1. Some examples in w hich f luor escent dyes have been found to inter act w ith uptake tr anspor ter s dir ectly as substrates or inhibi tor s. We do not include know n non-f luor escent substrates to w hich a f luor escent tag has been added (see e.g. [100-102]).

Dye Transporter Comments Reference Amiloride OCT2 (SLC22A2) Also a drug. Rhodamine 123 and 6G also [103]

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

served as substrates. 4’,6-diamidino-2-phenylindol (DAPI)

OCT1 (SLC22A1) Potently inhibited by desipramine. Also by various organophosphate pesticides.

[104]; [105]

DiBAC(4)3 Na+/HCO3- NBCe1-A

SLC4A4 Competes with 4,4’-Diisothiocyanatostilbene-2,2’-disulfonic acid

[106]

5-carboxyfluorescein OAT3 (SLC22A8 Very high Vmax [60] 6-carboxyfluorescein OAT1 (SLC22A6) [60]; [107] 2’,7’-dichlorofluorescein OATP1B1

(SLCO1B1) Good substrate [108]

4-(4-(Dimethylamino) styryl)- N-methylpyridinium (ASP+)

Dopamine transporter (SLC6A3)

[109; 110]

Noradrenaline transporter (SLC6A2)

[109; 111; 112]

Serotonin transporter SLC6A4

[109; 113]

Various monoamine transporters

[114]

OCT1/OCT2 (SLC22A1/2);

Seen as a model substrate [115]

Various OCT transporters

[116]

Other, unknown (non-OCT1/2) transporters with low affinity

[117]

Ethidium OCT1/2/3

(SLC22A1/2/3) Substrate [118]

FFN511 VMAT2 (SLC18A2) ‘False fluorescent neurotransmitter’ (i.e.

surrogate substrates) concept [43]

FFN54/246 SLC6A4, SLC18 ‘False’ fluorescent substrates for serotonin

and VMAT transporters. Potent inhibition by imipramine and citalopram

[44]

FFN270 SLC6A4, SLC18 Another example of a fluorescent false neurotransmitter

[119]

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Fluorescein SLCO1B1/3B1 Effective substrate; analysis of inhibitors [58] [59] OAT6 (SLC22A20) [60] SLC16A1, SLC16A4 [57] Many OATPs (SLCO

family) expressed in insect cells

[56]

Lucifer yellow Sodium-dependent

anion transporters Inhibited by probenecid [120; 121]

Rhodamine 123 OCT1/OCT2

(SLC22A1/2) Potent substrate [122]

Stilbazolium dyes Norepinephrine

transporter (SLC6) Dyes related to ASP+ [123]

Zombie Violet, Live/Dead Green, Cascade Blue, Alexa Fluor 405

OATP (SLCO) 1B1/1B3 and 2B1

All shown to be direct substrates, and uptake inhibited by known transporter inhibitors

[124]

Str u ctu r a l sim ila r ity (or th e a ssessm en t of p r oper ties based sim ply on an a lyzin g str u ctu r es) is an

elu sive con cep t (e.g. [125]), bu t a s ju dged by a stan da r d en cod in g (RDKit pa tter n ed) th er e is con sider ab le sim ila r ity in str u ctu r e betw een a lm ost a ll of ou r ch osen flu or oph or es an d a t least on e d r u g, w h eth er th is is ju dged by th eir descr ip tor - or fin ger pr in t-based pr oper ties (Figs 1-3), by obser va tion (Figs 4, 6), or (Fig 5) via th e QED [74] m easu r e.

Alth ou gh th er e is a m ove to ph en otyp ic scr een in g [126-129], m an y d r u gs w er e developed on th e basis of their abi l i ty to bind potently in vitro to a tar get of inter est. If the unbound molecule is confor mationally ver y f lexible, and the bound ver sion is not, binding necessar i ly involves a signif icant loss of entr opy. Potent binding (involving a signif icant loss in fr ee ener gy) of such a molecule w ould thus r equire a ver y lar ge enthalpic ter m. Consequently, i t is much easier to f ind potent binder s i f the binding can involve f lat (w hich implies ar omatic), confor mationally inf lexible planar str uctur es. Such r easoning pr esumably r ef lects the obser vation (Fig 5B) that dr ugs tend to have a low sp3 char acter , typically w ith a number of ar omatic r ings. Conjugated ar omatic r ings ar e also a major (physical and electr onic) str uctur e that al low flu or escen ce fr om or gan ic m olecu les [130-133], w ith gr ea ter π-bon d con ju ga tion m ovin g both absor ban ce an d flu or escen ce tow ar d th e r ed en d of th e spectr u m . Over a ll, th ese tw o sepa r a te r oles for a r om atic r esidu es, in low en tr opy of b in d in g an d in electr on ic str u ctu r e, p r ovide a p lau sib le explan a tion for m u ch of th e d r u g-liken ess of com m on flu or oph or es.

Wh ile th is stu dy u sed a com par a tively sm a ll set of flu or oph or es, in cr easin g th eir n u m ber can on ly in cr ease th e likelih ood of fin d in g a dr u g (or n a tu r a l p r odu ct) to w h ich th ey a r e seen to be sim ila r . Th is sa id , th is set of m olecu les p r ovides an excellen t sta r tin g poin t for th e developm en t of com petitive h igh -th r ou gh pu t a ssays of d r u g tr an spor ter activity.

CONCLUSIONS

An analysis of some 150 f luor ophor es in common usage in biological r esear ch has show n that a ver y gr eat many of them bear signif icant str uctur al simi lar i ties to mar keted dr ugs (and to natur al products).

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

Th is sim ila r ity h olds tr u e w h eth er th e an a lysis is don e u sin g str u ctu r es en coded as fin ger p r in ts or via ph ysico-ch em ica l descr ip tor s, by visu a l in spection , or via th e qu an tita tive estim a te of d r u g liken ess m easu r e. For an y given d r u g th er e is th u s likely to be a flu or oph or e or set of flu or oph or es th a t is best su ited to com petin g w ith it for u p take, an d th u s for deter m in in g by flu or im etr ic m eth ods th e QSAR for th e r elevan t tr an spor ter s. Th is sh ou ld p r ovide th e m ean s for r ap id an d con ven ien t com petitive an d tr an s-stim u la tion a ssays for scr een in g th e ab ility of d r u gs to en ter cells via SLCs.

SUPPLEMENTARY MATERIALS

The fol low ing supplementar y mater ials ar e avai lable onl ine in the f i le f luor ophor esSI.xlsx. Table S1: The l ist of al l the molecules and pr oper ties used in the pr esent analysis.

DATA AVAILABILITY

• Th e da ta set gen er a ted fr om (or an a lyzed in ) th e stu dy can be fou n d in th e Su pp lem en ta r y Excel sh eet en titled flu or oph or esSI.xlsx.

AUTHOR CONTRIBUTIONS DBK developed th e idea of deter m in in g flu or oph or e-d r u g sim ila r ity. SO’H w r ote an d r an th e m a jor ity of

th e w or kflow s an d cr ea ted ~tw o-th ir ds of th e visu a lisa tion s. Both au th or s con tr ibu ted to th e an a lysis of th e da ta an d to th e w r itin g of th e paper .

CONFLICTS OF INTEREST

Th e au th or s decla r e th a t th er e is n o con flict of in ter est.

FUNDING

Th is r esea r ch w as fu n ded by th e UK BBSRC (gr an t BB/P009042/1).

REFERENCES

[1] Ch a lfie, M. & Kain , S. (1998). Gr een Flu or escen t Pr otein : p r oper ties, app lica tion s, an d pr otocols. Wiley-Liss, New Yor k.

[2] Hemmilä, I . A. (1991). Applications of fluorescence in immunoassays. Wi ley, New Yor k. [3] Waggoner , A. S. (1990). Fluor escent Pr obes for Cytometr y. In Flow Cytometr y and Sor t ing (2nd Edit ion) (ed. M. R.

Melamed, T. Lindmo and M. L. Mendelsohn), pp. 209-225. Wiley-Liss Inc., New Yor k. [4] Kyr ych en ko, A. (2015). Usin g flu or escen ce for stu d ies of b iologica l m em br an es: a r eview . Methods Appl Fluoresc 3,

042003. [5] Lagor io, M. G., Cor don , G. B. & Ir iel, A. (2015). Review in g th e r elevan ce of flu or escen ce in b iologica l system s.

Photochem Photobiol Sci 14, 1538-59. [6] Spech t, E. A., Br aselm an n , E. & Pa lm er , A. E. (2017). A Cr itica l an d Com par a tive Review of Flu or escen t Tools for

Live-Cell Im agin g. Annu Rev Physiol 79, 93-117. [7] Sah l, S. J., Hell, S. W. & Jakobs, S. (2017). Flu or escen ce n an oscopy in cell b iology. Nat Rev Mol Cell Biol 18, 685-701. [8] Mavr akis, M., Pou r qu ie, O. & Lecu it, T. (2010). Ligh tin g u p developm en ta l m ech an ism s: h ow flu or escen ce im agin g

h er a lded a n ew er a . Development 137, 373-87. [9] Joh n son , I. (1998). Flu or escen t p r obes for livin g cells. Histochem J 30, 123-40. [10] Kolan ow ski, J. L., Liu , F. & New , E. J. (2018). Flu or escen t p r obes for th e sim u ltan eou s detection of m u ltip le an a lytes

in b iology. Chem Soc Rev 47, 195-208. [11] Zh an g, J., Cam pbell, R. E., Tin g, A. Y. & Tsien , R. Y. (2002). Cr ea tin g n ew flu or escen t p r obes for cell b iology. Nat Rev

Mol Cell Biol 3, 906-18. [12] Jian g, X., Wan g, L., Car r oll, S. L., Ch en , J., Wan g, M. C. & Wan g, J. (2018). Ch a llen ges an d Oppor tu n ities for

Sm all-Molecu le Flu or escen t Pr obes in Redox Biology Applica tion s. Antioxid Redox Signal 29, 518-540.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

[13] Win ter bou r n , C. C. (2014). Th e ch a llen ges of u sin g flu or escen t pr obes to detect an d qu an tify specific r eactive oxygen species in l iving cells. Biochim Biophys Acta 1840, 730-8.

[14] Davey, H. M. & Kel l, D. B. (1996). Flow cytometr y and cel l sor ting of heter ogeneous microbial populations: the impor tance of single-cell analysis. Microbiol. Rev. 60, 641-696.

[15] Shapir o, H. M. (2003). Practical Flow Cytometr y, 4th edit ion, 3r d edi tion. John Wiley, New Yor k. [16] Lavis, L. D. & Raines, R. T. (2008). Br ight ideas for chemical biology. ACS Chem Biol 3, 142-55. [17] Jindal, S., Yang, L., Day, P. J. & Kel l , D. B. (2019). Involvement of multiple inf lux and eff lux tr anspor ters in the

accumulation of cationic f luor escent dyes by Escher ichia coli. BMC Microbiol 19, 195; also bioRxiv 603688v1. [18] Kel l , D. B. (2019). Control of metaboli te eff lux in micr obial cell factor ies: cur r ent advances and futur e pr ospects. In

Fermentation microbiology and biotechnology, 4th Ed (ed. E. M. T. El-Mansi, J. Nielsen, D. Mousdale, T. Al lman and R. Car lson), pp. 117-138. CRC Pr ess, Boca Raton.

[19] Szakács, G., Vár adi , A., Özvegy-Laczka, C. & Sar kadi, B. (2008). The r ole of ABC tr anspor ter s in dr ug absor ption, distr ibution, metabol ism, excr etion and toxici ty (ADME-Tox). Drug Discov Today 13, 379-93.

[20] Schinkel, A. H. & Jonker , J. W. (2012). Mammalian dr ug eff lux tr anspor ter s of the ATP binding cassette (ABC) fami ly: an over view . Adv Drug Deliv Rev 64, 138-153.

[21] Tar l ing, E. J., de Aguiar Val l im, T. Q. & Edwar ds, P. A. (2013). Role of ABC tr anspor ter s in l ipid tr anspor t and human disease. Trends Endocr inol Metab 24, 342-350.

[22] Andr eoletti , P., Raas, Q., Gondcai l le, C., Cher kaoui-Malki , M., Tr ompier , D. & Savar y, S. (2017). Pr edictive Str uctur e and Topology of Per oxisomal ATP-Binding Cassette (ABC) Tr anspor ter s. Int J Mol Sci 18.

[23] Shar om, F. J. (2008). ABC multidr ug tr anspor ter s: str uctur e, function and r ole in chemor esistance. Pharmacogenomics 9, 105-27.

[24] Pahnke, J., Fr öhl ich, C., Paar mann, K., Kr ohn, M., Bogdanovic, N., År sland, D. & Winblad, B. (2014). Cerebr al ABC tr anspor ter -common mechanisms may modulate neur odegener ative diseases and depr ession in elder ly subjects. Arch Med Res 45, 738-43.

[25] Abuznait, A. H. & Kaddoumi, A. (2012). Role of ABC tr anspor ters in the pathogenesis of Alzheimer 's disease. ACS Chem Neurosci 3, 820-31.

[26] Lam, F. C., Liu, R., Lu, P., Shapir o, A. B., Renoir , J. M., Shar om, F. J. & Reiner , P. B. (2001). beta-Amyloid eff lux mediated by p-glycoprotein. J Neurochem 76, 1121-8.

[27] Molnar , J., Ocsovszki , I . & Pusztai , R. (2018). Amyloid-beta Inter actions w ith ABC Tr anspor ter s and Resistance Modif ier s. Anticancer Res 38, 3407-3410.

[28] Wi jnholds, J., Ever s, R., van Leusden, M. R., Mol, C. A. A. M., Zaman, G. J. R., Mayer , U., Bei jnen, J. H., van der Valk, M., Kr impenfor t, P. & Bor st, P. (1997). Incr eased sensi tivi ty to anticancer dr ugs and decr eased inf lammator y r esponse in mice lacking the multidr ug resistance-associated pr otein. Nat Med 3, 1275-9.

[29] Pr ates, R. A., Kato, I . T., Ribeir o, M. S., Tegos, G. P. & Hamblin, M. R. (2011). Inf luence of multidrug eff lux systems on methylene blue-mediated photodynamic inactivation of Candida albicans. J Antimicrob Chemother 66, 1525-32.

[30] Forster , S., Thumser , A. E., Hood, S. R. & Plant, N. (2012). Char acter ization of r hodamine-123 as a tr acer dye for use in in vitr o dr ug tr anspor t assays. PLoS One 7, e33253.

[31] Ivni tski-Steele, I ., Holmes, A. R., Lamping, E., Monk, B. C., Cannon, R. D. & Sklar , L. A. (2009). Identi f ication of Ni le r ed as a f luorescent substrate of the Candida albicans ATP-binding cassette tr anspor ter s Cdr 1p and Cdr 2p and the major faci l i tator super family tr anspor ter Mdr 1p. Anal Biochem 394, 87-91.

[32] Str ouse, J. J., Ivni tski-Steele, I ., Waller , A., Young, S. M., Per ez, D., Evangelisti , A. M., Ursu, O., Bologa, C. G., Car ter , M. B., Salas, V. M., Tegos, G., Lar son, R. S., Opr ea, T. I., Edwar ds, B. S. & Sklar , L. A. (2013). Fluor escent substr ates for f low cytometr ic evaluation of eff lux inhibi tion in ABCB1, ABCC1, and ABCG2 tr anspor ters. Anal Biochem 437, 77-87.

[33] Kel l, D. B. (2018). Control of metaboli te eff lux in micr obial cel l factor ies: cur rent advances and futur e pr ospects. OSF prepr ints, xg9jh.

[34] Szabó, E., Tür k, D., Telbisz, Á., Kucsma, N., Hor váth, T., Szakács, G., Homolya, L., Sar kadi, B. & Vár ady, G. (2018). A new f luor escent dye accumulation assay for par al lel measur ements of the ABCG2, ABCB1 and ABCC1 multidr ug tr anspor ter functions. PLoS One 13, e0190629.

[35] Gökirmak, T., Shipp, L. E., Campanale, J. P., Nickl isch, S. C. T. & Hamdoun, A. (2014). Tr anspor t in Technicolor : Mapping ATP-Binding Cassette Tr anspor ter s in Sea Ur chin Embr yos. Mol Reprod Dev 81, 778-793.

[36] Far del, O., Le Vee, M., Jouan, E., Denizot, C. & Par mentier , Y. (2015). Natur e and uses of f luor escent dyes for dr ug tr anspor ter studies. Exper t Opin Drug Metab Toxicol 11, 1233-1251.

[37] O'Hagan , S., Sw ain ston , N., Han dl, J. & Kell, D. B. (2015). A ‘r u le of 0.5′ for th e m etabolite-liken ess of appr oved ph ar m aceu tica l d r u gs. Metabolomics 11, 323-339.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

[38] O'Hagan , S. & Kell, D. B. (2015). Un der stan d in g th e fou n da tion s of th e str u ctu r a l sim ila r ities betw een m ar keted dr ugs and endogenous human metabol i tes. Front Pharmacol 6, 105.

[39] O'Hagan, S. & Kel l, D. B. (2016). MetMaxStr uct: a Tver sky-similar i ty-based str ategy for analysing the (sub)str uctur al simi lar i ties of dr ugs and endogenous metabol i tes. Front Pharmacol 7, 266.

[40] O'Hagan, S. & Kell , D. B. (2017). Analysis of dr ug-endogenous human metabol i te simi lar i ties in terms of their maximum common substructur es. J Cheminform 9, 18.

[41] Gr ündemann, D., Har l f inger , S., Golz, S., Geer ts, A., Lazar , A., Berkels, R., Jung, N., Rubber t, A. & Schömig, E. (2005). Discover y of the er gothioneine tr anspor ter . Proc Natl Acad Sci 102, 5256-61.

[42] O'Hagan, S. & Kel l , D. B. (2017). Consensus r ank or der ings of molecular f inger pr ints i l lustrate the ‘most genuine’ simi lar i ties between mar keted dr ugs and small endogenous human metabol i tes, but highl ight exogenous natur al pr oducts as the most impor tant ‘natur al ’ dr ug tr anspor ter substr ates. ADMET & DMPK 5, 85-125.

[43] Guber nator , N. G., Zhang, H., Staal, R. G., Moshar ov, E. V., Per eir a, D. B., Yue, M., Balsanek, V., Vadola, P. A., Mukher jee, B., Edwar ds, R. H., Sulzer , D. & Sames, D. (2009). Fluorescent false neur otr ansmitter s visual ize dopamine r elease fr om individual pr esynaptic ter minals. Science 324, 1441-4.

[44] Henke, A., Kovalyova, Y., Dunn, M., Dr eier , D., Guber nator , N. G., Dincheva, I ., Hwu, C., Sebej, P., Ansorge, M. S., Sulzer , D. & Sames, D. (2018). Towar d Ser otonin Fluor escent False Neur otr ansmitter s: Development of Fluor escent Dual Ser otonin and Vesicular Monoamine Tr anspor ter Substr ates for Visual izing Serotonin Neur ons. ACS Chem Neurosci 9, 925-934.

[45] Thiele, I ., Swainston, N., Fleming, R. M. T., Hoppe, A., Sahoo, S., Aur ich, M. K., Har aldsdottír , H., Mo, M. L., Rolfsson, O., Stobbe, M. D., Thor lei fsson, S. G., Agren, R., Böll ing, C., Bor del, S., Chaval i , A. K., Dobson, P., Dunn, W. B., Endler , L., Gor yanin, I ., Hala, D., Hucka, M., Hull , D., Jameson, D., Jamshidi , N., Jones, J., Jonsson, J. J., Juty, N., Keating, S., Nookaew , I., Le Novèr e, N., Malys, N., Mazein, A., Papin, J. A., Patel, Y., Pr ice, N. D., Selkov Sr ., E., Sigur dsson, M. I., Simeonidis, E., Sonnenschein, N., Smallbone, K., Sorokin, A., Beek, H. V., Weichar t, D., Nielsen, J. B., Wester hoff, H. V., Kel l, D. B., Mendes, P. & Palsson, B. Ø. (2013). A community-dr iven global r econstr uction of human metabol ism. Nat Biotechnol. 31, 419-425.

[46] Gu, J. Y., Gui, Y. S., Chen, L. R., Yuan, G., Lu, H. Z. & Xu, X. J. (2013). Use of Natur al Products as Chemical Libr ar y for Dr ug Discovery and Network Phar macology. PloS one 8, e62839.

[47] O'Hagan, S. & Kell , D. B. (2015). The KNIME wor kflow environment and i ts appl ications in Genetic Pr ogr amming and machine lear ning. Genetic Progr Evol Mach 16, 387-391.

[48] O'Hagan, S. & Kell , D. B. (2015). The appar ent per meabi l i t ies of Caco-2 cells to mar keted dr ugs: magnitude, and independence fr om both biophysical proper ties and endogenite similar i ties PeerJ 3, e1405.

[49] O'Hagan, S. & Kel l, D. B. (2018). Analysing and navigating natur al pr oducts space for gener ating small , diver se, but r epr esentative chemical l ibr ar ies. Biotechnol J 13, 1700503.

[50] O'Hagan, S. & Kel l, D. B. (2019). Generation of a small l ibr ar y of natur al pr oducts designed to cover chemical space inexpensively. Pharm Front 1, e190005.

[51] van der Maaten, L. & Hinton, G. (2008). Visual izing Data using t-SNE. J Machine Learning Res 9, 2579-2605. [52] Thiele, I ., Heinken, A. & Fleming, R. M. (2013). A systems biology appr oach to studying the r ole of microbes in

human health. Cur r Opin Biotechnol 24, 4-12. [53] Eisen, M. B., Spellman, P. T., Br own, P. O. & Botstein, D. (1998). Cluster analysis and display of genome-w ide

expr ession patter ns. Proc. Natl. Acad. Sci. 95, 14863-14868. [54] Bakkar , M. M., Har daker , L., March, P., Mor gan, P. B., Maldonado-Codina, C. & Dobson, C. B. (2014). The cel lular

basis for biocide-induced f luor escein hyper f luor escence in mammalian cel l cultur e. PLoS One 9, e84427. [55] Khan, T. F., Pr ice, B. L., Morgan, P. B., Maldonado-Codina, C. & Dobson, C. B. (2018). Cellular f luor escein

hyper f luor escence is dynamin-dependent and incr eased by Tetr onic 1107 tr eatment. Int J Biochem Cell Biol 101, 54-63.

[56] Patik, I ., Kovacsics, D., Német, O., Ger a, M., Vár ady, G., Stieger , B., Hagenbuch, B., Szakács, G. & Özvegy-Laczka, C. (2015). Functional expr ession of the 11 human Or ganic Anion Tr anspor ting Polypeptides in insect cel ls r eveals that sodium fluor escein is a gener al OATP substr ate. Biochem Pharmacol 98, 649-58.

[57] Sun, Y. C., Liou, H. M., Yeh, P. T., Chen, W. L. & Hu, F. R. (2017). Monocar boxylate Tr anspor ters Mediate Fluor escein Uptake in Corneal Epi thel ial Cel ls. Invest Ophthalmol Vis Sci 58, 3716-3722.

[58] De Br uyn, T., Fattah, S., Stieger , B., Augusti jns, P. & Annaer t, P. (2011). Sodium fluor escein is a pr obe substr ate for hepatic dr ug tr anspor t mediated by OATP1B1 and OATP1B3. J Pharm Sci 100, 5018-30.

[59] De Br uyn, T., van Westen, G. J., I jzerman, A. P., Stieger , B., de Witte, P., Augusti jns, P. F. & Annaer t, P. P. (2013). Str uctur e-based identi f ication of OATP1B1/3 inhibi tor s. Mol Pharmacol 83, 1257-67.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

[60] Tr u on g, D. M., Ka ler , G., Kh an delw al, A., Sw aan , P. W. & Nigam , S. K. (2008). Mu lti-level an a lysis of or gan ic an ion tr anspor ter s 1, 3, and 6 r eveals major di f fer ences in structur al deter minants of antivi r al discr imination. J Biol Chem 283, 8654-63.

[61] Pati l , S. A. & Moss, A. C. (2008). Balsalazide disodium for the tr eatment of ulcer ative col i tis. Exper t Rev Gastroenterol Hepatol 2, 177-84.

[62] Egesel, T., Ünsal, I ., Dikmen, G. & Bayr aktar , Y. (2002). The assessment of pancr eatic exocr ine function by benti r omide test in patients w ith chr onic por tal vein thr ombosis. Pancreas 25, 355-9.

[63] Singal, A. (2008). Butenafine and super f icial mycoses: cur r ent status. Exper t Opin Drug Metab Toxicol 4, 999-1005. [64] Blair , H. A. (2019). Tolvaptan: A Review in Autosomal Dominant Polycystic Kidney Disease. Drugs 79, 303-313. [65] Br own, N. & Jacoby, E. (2006). On scaffolds and hopping in medicinal chemistr y. Mini Rev Med Chem 6, 1217-29. [66] Gepper t, H. & Bajor ath, J. (2010). Advances in 2D f inger pr int simi lar i ty searching. Exper t Opin Drug Discov 5,

529-542. [67] Lamber th, C. (2018). Agr ochemical lead optimization by scaffold hopping. Pest Manag Sci 74, 282-292. [68] Mauser , H. & Guba, W. (2008). Recent developments in de novo design and scaffold hopping. Cur r Opin Drug Discov

Devel 11, 365-374. [69] Sun, H., Tawa, G. & Wallqvist, A. (2012). Classi f ication of scaffold-hopping appr oaches. Drug Discov Today 17,

310-24. [70] Zhao, H. (2007). Scaffold selection and scaffold hopping in lead gener ation: a medicinal chemistr y perspective.

Drug Disc Today 12, 149-55. [71] Das, A. M. (2017). Cl inical uti l i ty of ni tisinone for the tr eatment of her edi tar y tyr osinemia type-1 (HT-1). Appl Clin

Genet 10, 43-48. [72] Lock, E., Ranganath, L. R. & Timmis, O. (2014). The r ole of ni tisinone in tyr osine pathway disor der s. Cur r

Rheumatol Rep 16, 457. [73] Ranganath, L. R., Khedr , M., Milan, A. M., Davison, A. S., Hughes, A. T., Usher , J. L., Taylor , S., Loftus, N.,

Dar oszewska, A., West, E., Jones, A., Br iggs, M., Fisher , M., McCormick, M., Judd, S., Vinjamur i, S., Gr i f f in, R., Psar el l i , E. E., Cox, T. F., Sir eau, N., Di l lon, J. P., Devine, J. M., Hughes, G., Har rold, J., Bar ton, G. J., Jar vis, J. C. & Gallagher , J. A. (2018). Ni tisinone ar r ests ochronosis and decr eases r ate of pr ogr ession of Alkaptonur ia: Evaluation of the effect of ni tisinone in the United Kingdom National Alkaptonur ia Centr e. Mol Genet Metab 125, 127-134.

[74] Bicker ton, G. R., Paol ini , G. V., Besnar d, J., Mur esan, S. & Hopkins, A. L. (2012). Quanti fying the chemical beauty of dr ugs. Nat Chem 4, 90-8.

[75] Shultz, M. D. (2019). Two Decades under the Inf luence of the Rule of Five and the Changing Pr oper ties of Appr oved Or al Dr ugs. J Med Chem 62, 1701-1714.

[76] Lover ing, F., Bikker , J. & Humblet, C. (2009). Escape fr om f latland: incr easing satur ation as an appr oach to impr oving cl inical success. J Med Chem 52, 6752-6.

[77] Meyer s, J., Car ter , M., Mok, N. Y. & Brown, N. (2016). On the or igins of thr ee-dimensional i ty in dr ug-l ike molecules. Future Med Chem 8, 1753-67.

[78] Campbell , P. S., Jamieson, C., Simpson, I . & Watson, A. J. B. (2017). Pr actical synthesis of phar maceutically r elevant molecules enr iched in sp(3) char acter . Chem Commun (Camb) 54, 46-49.

[79] Blakemor e, D. C., Castr o, L., Chur cher , I ., Rees, D. C., Thomas, A. W., Wi lson, D. M. & Wood, A. (2018). Or ganic synthesis pr ovides oppor tuni ties to tr ansfor m dr ug discover y. Nat Chem 10, 383-394.

[80] Bostr öm, J., Br own, D. G., Young, R. J. & Keser ü, G. M. (2018). Expanding the medicinal chemistr y synthetic toolbox. Nat Rev Drug Discov 17, 709-727.

[81] Yuan, L., Lin, W., Zheng, K., He, L. & Huang, W. (2013). Far -r ed to near infrar ed analyte-r esponsive f luor escent pr obes based on or ganic f luor ophor e platfor ms for f luor escence imaging. Chem Soc Rev 42, 622-61.

[82] McInnes, L., Healy, J. & Melvi l le, J. (2018). UMAP: Unifor m Manifold Appr oximation and Pr ojection for Dimension Reduction. arXiv, 1802.03426v2.

[83] McInnes, L., Healy, J., Saul, N. & Gr oßber ger , L. (2018). UMAP: Unifor m Manifold Appr oximation and Pr ojection. J Open Source Software DOI 10.21105/joss.00861.

[84] Ivni tski-Steele, I ., Lar son, R. S., Lovato, D. M., Khawaja, H. M., Winter , S. S., Opr ea, T. I ., Sklar , L. A. & Edwar ds, B. S. (2008). High-thr oughput f low cytometr y to detect selective inhibi tor s of ABCB1, ABCC1, and ABCG2 tr anspor ter s. Assay Drug Dev Technol 6, 263-76.

[85] Tegos, G. P., Evangelisti , A. M., Str ouse, J. J., Ur su, O., Bologa, C. & Sklar , L. A. (2014). A high thr oughput f low cytometr ic assay platfor m tar geting tr anspor ter inhibi tion. Drug Disc Today Technol 12, e95-e103.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

[86] Win dt, T., Tóth , S., Pa tik, I., Sessler , J., Ku csm a, N., Szepesi, A., Zdr azil, B., Özvegy-Laczka , C. & Szakács, G. (2019). Identi f ication of anticancer OATP2B1 substr ates by an in vi tr o tr iple-f luor escence-based cytotoxici ty scr een. Arch Toxicol 93, 953-964.

[87] Gautier , J., Munnier , E., Souce, M., Chour pa, I . & Douziech Eyr ol les, L. (2015). Analysis of doxor ubicin distr ibution in MCF-7 cel ls tr eated w ith dr ug-loaded nanopar ticles by combination of two f luor escence-based techniques, confocal spectr al imaging and capi l lar y electr ophor esis. Anal Bioanal Chem.

[88] Khader , H., Solodushko, V., Al-Mehdi, A. B., Audia, J. & Fouty, B. (2014). Over lap of Doxycycl ine Fluor escence w ith that of the Redox-Sensi tive Intr acel lular Repor ter r oGFP. Journal of Fluorescence 24, 305-311.

[89] Motlagh, N. S. H., Parvin, P., Ghasemi, F. & Atyabi, F. (2016). Fluor escence pr oper ties of sever al chemother apy dr ugs: doxor ubicin, pacl i taxel and bleomycin. Biomed Opt Express 7, 2400-6.

[90] Baldini , G., Dogl ia, S., Dolci , S. & Sassi , G. (1981). Fluor escence-deter mined pr efer ential binding of quinacr ine to DNA. Biophys J 36, 465-77.

[91] Nguyen, M., Mar cellus, R. C., Roulston, A., Watson, M., Ser fass, L., Mur thy Madir aju, S. R., Goulet, D., Vial let, J., Belec, L., Bi l lot, X., Acoca, S., Pur isima, E., Wiegmans, A., Cluse, L., Johnstone, R. W., Beaupar lant, P. & Shor e, G. C. (2007). Small molecule obatoclax (GX15-070) antagonizes MCL-1 and over comes MCL-1-mediated r esistance to apoptosis. Proc Natl Acad Sci U S A 104, 19512-7.

[92] Stamelos, V. A., Fisher , N., Bamr ah, H., Voisey, C., Pr ice, J. C., Far rel l , W. E., Redman, C. W. & Richar dson, A. (2016). The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkal inization. PLoS One 11, e0150696.

[93] Pautke, C., Vogt, S., Kr eutzer , K., Haczek, C., Wexel, G., Kolk, A., Imhoff, A. B., Zi tzelsber ger , H., Milz, S. & Tischer , T. (2010). Char acter ization of eight di f fer ent tetr acycl ines: advances in f luor escence bone label ing. J Anat 217, 76-82.

[94] Bur ke, T. G., Malak, H., Gr yczynski, I ., Mi, Z. & Lakow icz, J. R. (1996). Fluor escence detection of the anticancer dr ug topotecan in plasma and whole blood by two-photon exci tation. Anal Biochem 242, 266-70.

[95] Yonezawa, A. & Inui , K. (2013). Novel r iboflavin tr anspor ter fami ly RFVT/SLC52: identi f ication, nomenclatur e, functional char acter ization and genetic diseases of RFVT/SLC52. Mol Aspects Med 34, 693-701.

[96] Zhang, S., Sakuma, M., Deor a, G. S., Levy, C. W., Klausing, A., Breda, C., Read, K. D., Edl in, C. D., Ross, B. P., Wr ight Muelas, M., Day, P. J., O'Hagan, S., Kel l, D. B., Schwar cz, R., Leys, D., Heyes, D. J., Gior gini , F. & Scr utton, N. S. (2019). A br ain-per meable inhibi tor of the neur odegener ative disease tar get kynur enine 3-monooxygenase pr events accumulation of neur otoxic metabol i tes. Commun Biol 2, 271.

[97] Liu, Q., Liu, Y., Guo, M., Luo, X. & Yao, S. (2006). A simple and sensi tive method of nonaqueous capi l lar y electr ophor esis w ith laser -induced native f luor escence detection for the analysis of cheler ythr ine and sanguinar ine in Chinese her bal medicines. Talanta 70, 202-7.

[98] Duval, R. & Duplais, C. (2017). Fluorescent natur al pr oducts as pr obes and tr acer s in biology. Nat Prod Rep 34, 161-193.

[99] Taniguchi, M. & Lindsey, J. S. (2018). Database of Absor ption and Fluorescence Spectr a of >300 Common Compounds for use in PhotochemCAD. Photochem Photobiol 94, 290-327.

[100] Bednar czyk, D., Mash, E. A., Aavula, B. R. & Wr ight, S. H. (2000). NBD-TMA: a novel f luor escent substr ate of the per i tubular or ganic cation tr anspor ter of r enal pr oximal tubules. Pflugers Arch 440, 184-92.

[101] Stone, M. R. L., Butler , M. S., Phetsang, W., Cooper , M. A. & Blaskovich, M. A. T. (2018). Fluor escent Antibiotics: New Resear ch Tools to Fight Antibiotic Resistance. Trends Biotechnol 36, 523-536.

[102] Jiang, M., Li , H., Johnson, A., Kar asawa, T., Zhang, Y., Meier , W. B., Taghizadeh, F., Kachelmeier , A. & Steyger , P. S. (2019). Inflammation up-r egulates cochlear expr ession of TRPV1 to potentiate dr ug-induced hear ing loss. Sci Adv 5, eaaw1836.

[103] Ugwu, M. C., Pel is, R., Esimone, C. O. & Agu, R. U. (2017). Fluor escent organic cations for human OCT2 tr anspor ters scr eening: uptake in CHO cel ls stably expressing hOCT2. ADMET & DMPK 5, 135-145.

[104] Yasujima, T., Ohta, K., Inoue, K. & Yuasa, H. (2011). Char acter ization of human OCT1-mediated tr anspor t of DAPI as a f luor escent pr obe substr ate. J Pharm Sci 100, 4006-12.

[105] Chedik, L., Br uyer e, A. & Far del, O. (2018). Inter actions of or ganophosphor us pesticides w ith solute car r ier (SLC) dr ug tr anspor ter s. Xenobiotica, 1-12.

[106] Liu, X., Wi l l iams, J. B., Sumpter , B. R. & Bevensee, M. O. (2007). Inhibi tion of the Na/bicar bonate cotr anspor ter NBCe1-A by diBAC oxonol dyes r elative to ni f lumic acid and a sti lbene. J Membr Biol 215, 195-204.

[107] Zou, L., Stecula, A., Gupta, A., Pr asad, B., Chien, H. C., Yee, S. W., Wang, L., Unadkat, J. D., Stahl, S. H., Fenner , K. S. & Giacomini , K. M. (2018). Molecular Mechanisms for Species Differ ences in Or ganic Anion Transpor ter 1, OAT1: Implications for Renal Dr ug Toxici ty. Mol Pharmacol 94, 689-699.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

[108] Izu m i, S., Nozaki, Y., Kom or i, T., Taken aka , O., Maeda , K., Ku su h ar a , H. & Su giyam a, Y. (2016). In vestiga tion of Flu or escein Der iva tives as Su bstr a tes of Or gan ic An ion Tr an spor tin g Polypeptide (OATP) 1B1 To Develop Sensitive Fluor escence-Based OATP1B1 Inhibi tion Assays. Mol Pharm 13, 438-48.

[109] Schwar tz, J. W., Blakely, R. D. & DeFelice, L. J. (2003). Binding and tr anspor t in nor epinephr ine tr anspor ter s. Real-time, spatial ly r esolved analysis in single cells using a f luor escent substr ate. J Biol Chem 278, 9768-77.

[110] Inyushin, M. U., Ar encibia-Albi te, F., de la Cr uz, A., Vazquez-Tor r es, R., Colon, K., Sanabr ia, P. & Jimenez-River a, C. A. (2013). New method to visual ize neur ons w ith DAT in sl ices of r at VTA using f luor escent substr ate for DAT, ASP+. J Neurosci Neuroeng 2, 98-103.

[111] Schwar tz, J. W., Novar ino, G., Piston, D. W. & DeFelice, L. J. (2005). Substr ate binding stoichiometr y and kinetics of the nor epinephr ine tr anspor ter . J Biol Chem 280, 19177-84.

[112] Haunsø, A. & Buchanan, D. (2007). Phar macological char acter ization of a f luor escent uptake assay for the nor adr enal ine tr anspor ter . J Biomol Screen 12, 378-84.

[113] Oz, M., Libby, T., Kivel l, B., Jal igam, V., Ramamoor thy, S. & Shippenber g, T. S. (2010). Real-time, spatial ly r esolved analysis of ser otonin tr anspor ter activi ty and r egulation using the f luor escent substr ate, ASP+. J Neurochem 114, 1019-29.

[114] Zwar tsen, A., Ver boven, A. H. A., van Kleef, R., Wi jnolts, F. M. J., Wester ink, R. H. S. & Hondebr ink, L. (2017). Measur ing inhibi tion of monoamine r euptake tr anspor ter s by new psychoactive substances (NPS) in r eal-time using a high-thr oughput, f luor escence-based assay. Toxicol In Vitr o 45, 60-71.

[115] Kido, Y., Matsson, P. & Giacomini, K. M. (2011). Profi l ing of a pr escr iption dr ug l ibr ar y for potential r enal dr ug-dr ug inter actions mediated by the or ganic cation tr anspor ter 2. J Med Chem 54, 4548-58.

[116] Salomon, J. J., Endter , S., Tachon, G., Falson, F., Buckley, S. T. & Ehr har dt, C. (2012). Tr anspor t of the f luor escent or ganic cation 4-(4-(dimethylamino)styr yl)-N-methylpyr idinium iodide (ASP+) in human r espir ator y epi thel ial cel ls. Eur J Pharm Biopharm 81, 351-9.

[117] Rytting, E., Br yan, J., Southar d, M. & Audus, K. L. (2007). Low-aff ini ty uptake of the f luorescent organic cation 4-(4-(dimethylamino)styr yl)-N-methylpyr idinium iodide (4-Di-1-ASP) in BeWo cel ls. Biochem Pharmacol 73, 891-900.

[118] Lee, W. K., Reichold, M., Edemir , B., Ciar imboli , G., War th, R., Koepsel l, H. & Thévenod, F. (2009). Organic cation tr anspor ter s OCT1, 2, and 3 mediate high-aff ini ty tr anspor t of the mutagenic vi tal dye ethidium in the kidney pr oximal tubule. Am J Physiol Renal Physiol 296, F1504-13.

[119] Dunn, M., Henke, A., Clar k, S., Kovalyova, Y., Kempadoo, K. A., Kar pow icz, R. J., Jr ., Kandel, E. R., Sulzer , D. & Sames, D. (2018). Designing a nor epinephr ine optical tr acer for imaging individual nor adr ener gic synapses and their activi ty in vivo. Nat Commun 9, 2838.

[120] Maser eeuw , R., Moons, M. M., Toomey, B. H., Russel, F. G. M. & Mi l ler , D. S. (1999). Active luci fer yel low secr etion in r enal pr oximal tubule: evidence for or ganic anion tr anspor t system cr ossover . J Pharmacol Exp Ther 289, 1104-11.

[121] de Gier , R. P. E., Feitz, W. F. J., Maser eeuw , R., Wouter se, A. C., Smits, D. & Russel, F. G. M. (2003). Anionic and cationic dr ug secr etion in the isolated per fused r at kidney after neonatal surgical induction of ur eter ic obstr uction. BJU Int 92, 452-8.

[122] Jouan, E., Le Vee, M., Denizot, C., Da Violante, G. & Far del, O. (2014). The mitochondr ial f luor escent dye r hodamine 123 is a high-aff ini ty substr ate for or ganic cation tr anspor ters (OCTs) 1 and 2. Fundam Clin Pharmacol 28, 65-77.

[123] Wilson, J. N., Brown, A. S., Babinchak, W. M., Ridge, C. D. & Walls, J. D. (2012). Fluor escent sti lbazol ium dyes as pr obes of the nor epinephr ine tr anspor ter : str uctur al insights into substr ate binding. Org Biomol Chem 10, 8710-9.

[124] Patik, I ., Székely, V., Német, O., Szepesi , Á., Kucsma, N., Vár ady, G., Szakács, G., Bakos, É. & Özvegy-Laczka, C. (2018). Identi f ication of novel cel l-imper meant f luor escent substr ates for testing the function and dr ug inter action of Or ganic Anion-Tr anspor ting Polypeptides, OATP1B1/1B3 and 2B1. Sci Rep 8, 2630.

[125] Lajiness, M. S., Maggior a, G. M. & Shanmugasundar am, V. (2004). Assessment of the consistency of medicinal chemists in r eview ing sets of compounds. J Med Chem 47, 4891-6.

[126] Kel l, D. B. (2013). Finding novel phar maceuticals in the systems biology er a using multiple effective dr ug tar gets, phenotypic scr eening, and know ledge of tr anspor ter s: wher e dr ug discover y went w r ong and how to f ix i t. FEBS J 280, 5957-5980.

[127] Kel l, D. B., Wr ight Muelas, M., O'Hagan, S. & Day, P. J. (2018). The role of dr ug tr anspor ter s in phenotypic scr eening. Drug Target Review 4, 16-19.

[128] Pr ior , M., Chir uta, C., Cur r ais, A., Goldber g, J., Ramsey, J., Dargusch, R., Maher , P. A. & Schuber t, D. (2014). Back to the futur e w ith phenotypic scr eening. ACS Chem Neurosci 5, 503-13.

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint

[129] Sw inney, D. C. (2014). Oppor tuni ties for phenotypic scr eening in dr ug discovery. Drug Disc Wor ld 15, 33-42. [130] Yam agu ch i, Y., Matsu bar a , Y., Och i, T., Wakam iya , T. & Yosh ida , Z. (2008). How th e π con ju ga tion len gth a ffects

th e flu or escen ce em ission efficien cy. J Am Chem Soc 130, 13867-9. [131] Lavis, L. D. & Ra in es, R. T. (2014). Br igh t bu ild in g b locks for ch em ica l b iology. ACS Chem Biol 9, 855-66. [132] Zam bian ch i, M., Di Mar ia , F., Cazza to, A., Gigli, G., Piacen za , M., Della Sa la , F. & Bar bar ella , G. (2009).

Micr ow ave-assisted syn th esis of th ioph en e flu or oph or es, labelin g an d m u ltilabelin g of m on oclon a l an tibod ies, an d lon g la stin g sta in in g of fixed cells. J Am Chem Soc 131, 10892-900.

[133] Di Mar ia , F., Pa lam à , I. E., Bar on cin i, M., Bar b ier i, A., Bon gin i, A., Bizza r r i, R., Gigli, G. & Bar bar ella , G. (2014). Live cell cytop lasm sta in in g an d selective labelin g of in tr acellu la r p r otein s by n on -toxic cell-per m ean t th ioph en e flu or oph or es. Org Biomol Chem 12, 1603-10.

How to cite th is a r ticle:

O’Hagan S, Kell DB. Str u ctu r a l sim ila r ities betw een som e com m on flu or oph or es u sed in b iology an d m ar keted

dr u gs, en dogen ou s m etabolites, an d n a tu r a l p r odu cts. Ph ar m Fr on t. 2019, su bm itted for r eview .

.CC-BY 4.0 International licenseauthor/funder. It is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the. https://doi.org/10.1101/834325doi: bioRxiv preprint