Downloaded by guest on September 28, 2021 VO—ugse n doae yLc ui 1,12)— (11, Turin Luca by advocated and (VTO)—suggested 10). (9, observed been light- also have in inactivated proteins changes sensitive and struc- conformational activated the photon-induced Additionally, examining the by of several pathways tures through sidechain conformations peptide between may proposed agonist interchange the molecular the how used into assist insights work structural additional provide to An dynamics acti- with (5–7). determined receptors spectroscopy experimentally isotopic-tagged NMR has through papers states of proteins. vated/inactivated G series to e.g., couple recent cell, that the A within loops molecules intracellular signaling to the appropriate leads of change structural conformations conformational This altered bundle. a helical in the the resulting within into change site, moving (4). binding agonist G membrane orthosteric appropriate an (interior) the protein’s by of of activated integrity activation are the GPCRs through maintaining interior while sig- cellular proteins, ligand the joint pro- extracellular to transmembrane the from nals Axel 7-helical to communication Richard are led facilitating to GPCRs teins, ORs Medicine (3) and of Buck. Linda Physiology cloning and in hippocampus the Prize the and Nobel discovery and 2004 to The cortex, information 2). orbitofrontal conveys (1, and the encoded amygdala, the is the within it responses where (GPCRs)—located mediating bulb, and receptors olfactory epithelium (ORs)— protein-coupled nasal receptors the G olfactory within of of activation subclass by a occur olfaction to human known Specifically, kingdoms. is and clades many of isms O tunneling electron protein the of members examined acti- the the for or ligands 5-HT affinity selected binding the the by vation either affected selec- that deuteration found was tive evidence No isotopologues. deuterated 5-HT tive the 2,5-dimethoxy-4-iodoamphetamine of and receptors, members (DOI) serotonin for using agonists of performed well-characterized was class acti- test of receptor This pair and flux. a affinity calcium receptor by by measured both vation idea, this of viability (2015) ner- al. (2013) (2012) SJ OS et central Oh June HK, the recep- HK, Chee with protein-coupled [Chee 11(4):282–288; associated (CNS). G system those nonolfactory vous including the- to (GPCRs), this applied that tors be offered may been of an has some ory papers, portion evidence with of series theoretical inelastic agonist recent suggestive a the an In energy. accept electron’s of tunneling to presence the mechanism state the tunneling vibrational through electron appropriate mediated protein-coupled inelastic is G an that olfactory by of occurs activation receptors the the- This that attention. proposes 2016) garnered which 6, ory has November by activated review method for are the (received proteins 2017 concerning olfactory 14, theory April approved alternative and NY, an York, Recently, New University, Rockefeller The Vosshall, B. Leslie by Edited c a activation Hoehn D. receptor Ross protein-coupled G of vibrational theory generalized the of evaluation Experimental www.pnas.org/cgi/doi/10.1073/pnas.1618422114 ogeLsAgls eie A921 and 90291; CA Venice, Angeles, Los Google eateto hmsr,Pru nvriy etLfyte N47907; IN Lafayette, West University, Purdue Chemistry, of Department eety nieaino h irtoa hoyo olfaction of theory vibrational the of iteration an Recently, 2 nipratifraingteigtcnqefrorgan- for technique information-gathering important processes—is an sensory chemo-sensitive other lfaction—and eetrclass. receptor | ehns faction of mechanism N, eoisInform Genomics dmtylsraie(A-7,adterrespec- their and (DAM-57), -dimethyllysergamide a ai .Nichols E. David , | pharmacology 02:2–3] een ets the test we Herein, 10(2):128–132]. b,1 d onD McCorvy D. John , ESLett FEBS at eIsiue at e M87501 NM Fe, Santa Institute, Fe Santa | oii eetr(8). receptor µ-opioid unu biology quantum eoisInform Genomics 589(4):548–552; | b atu Neven Hartmut , b eateto hraooy nvriyo ot aoia hplHl,N 27514; NC Hill, Chapel Carolina, North of University Pharmacology, of Department 2 t fteset stp xhnetsso h T oue on focused VTO the of tests exchange qual- Isotope the or scent. intensity 1 the the chemistry—may either of of its ity the alteration maintaining an alter as while itself to molecule manifest ability (33– a the GPCRs of non-OR provides vibrations into exchange—which expanded Isotope and 36). contin- (32), (12), ORs perception within olfactory ued with characteristics spectra modern correlated vibrational VTO the the the of evaluate of to justifications used iso- Initial been and VTO. 31)—has 18) (11, (11, exchange blending methods—odor tope these (20, exchange of isotope each and 27–30); 26) (25, blending odor including VTO, and results. humans controversial both and with mixed 22–24). 16–18, provided more theory (14, have Additionally, this theory subjects 15). this of insect (13, disprove examinations tests to modern includ- addressed sensory theory, been controlled this have through for mixing, tests odor proposed cas- ing the signal at-receptor of direct intracellular lacks Several and the contentious evidence. is to both leading theory This change GPCR, cade. conformation olfactory the the instigates Hypothet- the of transfer junction. the that electron (ET) suggests this within electron-tunneling theory ically, an available this as of is behaves of incarnation OR excited, photoexcitation modern activation thermally for The the entirely body. mechanism for were no responsible vibrations vibrations as These exactly molecular protein. were the the agonist that of the suggested vibrations of Dyson recep- molecular olfactory olfactant. to—the the Wright sensitive the of and performed—or activation (20), is the Wright tor is where (19), (21), VTO Dyson Serenius the clas- by and more proposed of the of theory incarnation reincarnation sical contemporary this a mechanism. is nonphoton-based of theory and Turin’s novelty detrac- nonthermal- its and the to (13–16) ascribable 18); supporters both (17, gained tors has and arisen has 1073/pnas.1618422114/-/DCSupplemental. at online information supporting contains article This Submission. 1 Direct PNAS a is article This interest. of conflict no declare and authors data; The analyzed paper. the S.K. wrote and S.K. H.N., and J.D.M., D.E.N., D.E.N., R.D.H., R.D.H., research; performed J.D.M. and uhrcnrbtos ...... n ..dsge eerh ... D.E.N., R.D.H., research; designed S.K. and H.N., D.E.N., R.D.H., contributions: Author purdue.edu. owo orsodnemyb drse.Eal aspru.d rdrdave@ or [email protected] Email: addressed. be may correspondence whom To H→ nlsso gns eairbtenisotopologues. between comparative behavior agonist a of through analysis performed was 2,5- Invalidation the agonists. both with and and h5-HT2 dimethoxy-4-iodoamphetamine family the at receptor done serotonin was This CNS receptors. protein-coupled recep- G of nonolfactory family a tor at activation and recep- affinity olfactory measuring of tors activation the from for obtained theory predictions vibrational Turin’s theory comparing vibrational by the activation of protein iteration of present the test we Herein, Significance hr aebe eea et ftepeiu trtoso the of iterations previous the of tests several been have There 2 xhne although exchange, H c n ar Kais Sabre and , PNAS | a 0 2017 30, May a,d,1 13 xhnehsbe suggested been has exchange C | N, o.114 vol. www.pnas.org/lookup/suppl/doi:10. -dimethyllysergamide | o 22 no. | 5595–5600

BIOPHYSICS AND COMPUTATIONAL BIOLOGY a CH3 receptor used as a standard ligand for studying the pharmacol- ABb N CH3 ogy of 5-HT2A receptors and is widely used as a radioligand to measure expression and affinity of ligands at 5-HT2A recep- O tors, particularly in brain tissues. (R)-DOI is reported to have CH 3 psychedelic effects somewhat similar to those of lysergic acid OCH3 N H16 H17 diethylamide (LSD) (56). A quintessential hallucinogenic ligand H H for the 5-HT2A receptor is LSD; a highly related (non-Schedule CH3 1) molecule is generated by substituting the diethyl amide with a dimethyl amide, creating N,N-dimethyllysergamide (DAM-57).

NH2 DAM-57 is an assumed serotonergic psychedelic with limited H18 hallucinogenic capacity and very mild autonomic stimulation in I humans at dosages of ∼100µg (57). The organization of this paper is as follows: Theoretical Pre- dictions gives a brief explanation of the theoretical predictions OCH3 N for (R)-DOI at members of the 5-HT2 serotonin receptor class H according to Turin’s theory, while making direct reference to Fig. 1. (A) Structure of the (R)-DOI molecule. In blue text, the atomic a previous work concerning similar predictions for DAM-57; indexes for some specific sidechain hydrogen atoms initially considered Experimental Procedures briefly introduces methods of experi- for deuteration are shown. Additional sites considered for deuteration are mental analysis for determining affinity; Results and Discussion the hydrogens of the two methoxy groups. (B) Structure of the DAM- provides a discussion of the experimental results and a compar- 57 molecule. In blue text, each methyl amide carbon where deuteration ison with theoretical predictions; and finally, in Conclusion we exchange was undertaken is denoted as either carbon “a” or “b.” provide concluding remarks. Theoretical Predictions (37). Unfortunately, measuring either quality or intensity Turin—within the contemporary VTO—hypothesized that the changes by sensory studies has presented itself as inconclusive active site of the GPCR [specifically an OR, although later works and difficult to quantify. considered generalizing this hypothesis (18, 36, 58)] acts as an Recently, several recent studies—both in vitro and in vivo— ET junction (11). According to the theory, an electron emerges have been conducted at a more physiological level. Block et al. from a donation site—likely a metal atom acting as a cofac- (17) studied the activation of several ORs (importantly, tor (11, 31), redox chemistry (59), or peptide sidechain (11, 60) OR5AN1 and MOR244-3) by both muscone and cyclopen- capable of oxidation—and traverses the active site to an accep- tadecanone; neither receptor displayed a significant differen- tor site, which is likely a specific motif or residue sidechain. As tial response to isotopologues of the musk odorants, suggest- the electron traverses the active site, it may undertake several ing failure of the VTO at mammalian ORs. However, criticism paths: (i) elastic tunneling, where no energy is lost or gained by of this work has arisen on grounds of lacking the in situ envi- the electron; (ii) inelastic tunneling (IET), where the electron ronment (odorant binding proteins, cofactors, etc.) and possibly may donate or accept a quantum of energy during transfer; and being too specific with respect to the repertoire of examined ORs (iii) subsequently higher ordered inelastic processes (61–63). The (23, 24, 38, 39). Supporting previous behavioral studies (14, 16), hypothesized presence of a possible metal cofactor site—acting Drimyl et al. (23) and Paoli et al. (24) studied direct electro- to assist either in binding or in a later activation step—at ORs, physical responses induced by odorant detection at the antennae GPCRs, and non-GPCR chemokine receptors is supported by and glomeral lobe of several Drosophila species and Apis mel- altered behavioral response (64–66), physiological response (66– lifera, respectively. These findings show unique spatial–temporal 68), theory (69, 70), and in vitro observations (70–79). responses with respect to families of isotopologues. Two things should be further noted with respect to these in situ insect stud- ies: (i) Despite attempts at compensation, these results may not be entirely independent of perireceptor effects (including trans- port, enzymes, and extracellular vestibule). (ii) Insect ORs are ionotropic receptors (IRs) and not GPCRs; each class of recep- tors (GPCRs vs. IRs) shows specific evolutionary benefits (broad responsiveness vs. speed of detection and processing) (40, 41). Herein, we exploit the fact that ORs are a subclass of the broader GPCR family with highly conserved sequences and structural motifs. The nonexceptionalism of ORs within the broader class of GPCRs was previously discussed within the con- text of the VTO (18) and is highlighted by Barwich (42), who asserts ORs are a model system for the GPCRs within neuro- biology. Furthermore, ORs maintain up to 40% genetic similar- ity with rhodopsin (43). Additionally, ORs appear within areas of mammalian corpus that have no olfactory capacity (44–55). We therefore hypothesize that due to functional and morpholog- ical similarities, if ORs are activated through an ET mechanism, other GPCRs share the same fundamental mechanism. Examina- tion of another (better characterized) GPCR subclass may pro- Fig. 2. Plots of the tunneling probability as a function of energy, P(E), for vide insight into the possibility of the proposed ET mechanism. various deuterated analogues of the DOI molecule. (Top Left) In orange, The serotonin 5-HT2 receptor class, notably a primary target 2 2 H16 deuterated DOI; (Top Right) in green H18 deuterated DOI; (Bottom 2 2 for hallucinogenic compounds, was selected as the main test for Left) in magenta, H16 and H17 deuterated DOI; and (Bottom Right) in cyan, 2 2 2 the contemporary VTO. (R)-2,5-dimethoxy-4-iodoamphetamine H16, H17, and H18 deuterated DOI. In all plots, blue is the all-protium DOI (R-DOI) is a well-characterized agonist for the serotonin 5-HT2A tunneling spectrum.

5596 | www.pnas.org/cgi/doi/10.1073/pnas.1618422114 Hoehn et al. Downloaded by guest on September 28, 2021 Downloaded by guest on September 28, 2021 oh tal. et Hoehn mode internal an (to that energy such capa- ligand) of is bound quantum electron a specific of the a if it losing However, process, of site. tunneling ble acceptor elastic available an no undergo energy, has of to quantum attempts a electron by an offset are energies acceptor and a with out carried be could experiment cm this effect. 1,600–1,800 dou- detectable that at the indicating peaks that active 50%, Note possible roughly groups. the the methoxy to depletes aromatic deuteration respect both ble with or molecule one (R)-DOI of the deuteration of analogues deuterated various 3. Fig. hoeia td fsvrllgnsblnigt h aiyof family the to belonging ligands several of study theoretical was and (84). transfer al. et assist Bittner interac- to by This modes used others. recently Raman among permits (63), potential al. Adkins et tion and Kirtley Phillips (61), and Jaklevic and (62), Lambe vibrat- by each dipole—described of ing orientation electron–dipole the fundamental on theoretical emphasis the future interaction—placing impor- include Furthermore, are (59). should processes evi- activation investigations oxidative is GPCR other there in as and tant pos- species, NADPH the molecular that including is perireceptor dence cofactors OR of all the effects for of mechanism sible account structure models delivery complete and the electron known until the addressed be of cannot reliability (82). the environmental- signaling vibrational that Evaluating the shown enhance have could dissipation studies electron induced such the and with bath (80), vibrational transfer a previously were coupling environment through reorganization the addressed into to the leaking conform that density can electron and OR of transfer, an the for (λ) on energy have effect fluctuations but small nonzero—all dynamic very a that a showing have ligand, receptor concerns, the the on these of negligible—effect of effects several binding Recently, addressed the mechanism. that (81) delivery al. electron et unreliable Reese during an couplings and electron ET, protein possible excluding inappropriate system, on the fluctuations include dynamic criticisms considering not these energies, reorganization VTO; the to approaches (83). effects chiral and (80–82), rates effects transfer receptor charge 80), including (60, system, spe- the for of account considerations to cific several undertaken hypothesis, been have Turin’s to expansions within process theoretical IET Working an and protein. in orientation the assisting correct of activate the capable with mode molecule vibrational site a a active have Thus, the protein. into fit the both activating must undertaken, be can fer ihntehpteie rti-ae Tjnto,tedonor the junction, ET protein-based hypothesized the Within ei h otx fTrnsVOpeiul odce a conducted VTO—previously Turin’s of context the We—in modeling current of criticisms several gave (17) al. et Block lt ftetneigpoaiiya ucino nry () for P(E), energy, of function a as probability tunneling the of Plots E acceptor − λ  E donor 1 clml Concerns kcal/mol. ∆E = h trans- the , ∆E −1 If . by (i ausaeepesda nanomolar. as expressed are values *(Ki) R-d o 5-HT for h5-HT Ligand hexadeutero its receptors and human (R)-DOI cloned of at affinities isotopologue Binding 1. Table relative the elec- and (displacement), an dipole and the of dipole) size (oscillating (ii the on mode. mode dependent vibrational vibrational is specific tron a a between of coupling quanta of energy energy specific of a shifting (i in effects: in interrelated probability two to tunneling attributable is associated range. region energy the completing this of electron within an depletion modes of vibrational This by probability assisted lower transfer prob- much tunneling tunneling a a the in Reducing result 86). would (85, typi- ability effect a isotope than larger derived be kinetically would cal effect Pro-protium this additionally 3, evident; experimentally Fig. Within d DOI. blue, an of for in ring (PDF) shown aromatic is function (d DOI the distribution one on of probability groups deuteration the subsequent methoxy plotted the during have IET we the 3 Fig. In Procedures Experimental leading 86), findings. (85, these itself of disregard 8–10% further be a a may highlighting to that possibly effect vibrations, to isotope the alter- attributable kinetic Additionally, changes be effects. isotopes could isotope of it kinetic ation as of smaller causes convincing, anything normal be efficacy, the not of this may terms Although 10% in 10%. than difference roughly of substantial decrease a a is is the associated schemes in current, these alteration tunneling with the The thus region. tun- and energy the deuteration probability, target on tunneling no the effect 2, large in Fig. a spectra in 2, produces neling sidechain seen Fig. alkyl be in the can is of plot As and scheme each compound comparison. Within abundance a natural as 2. the given Fig. denotes in curve tunnel- seen blue the the on be deuteration can of spectrum effects ing The efficacy. agonist change sufficient the present found in would we analogue DOI iso- deuterium of such it kinetic sidechain no that pedestrian ethyl that the degree the deuterating to enough By attributable high effects. solely tope a be to to receptor vari- unlikely the possible is at the efficacy maximizing in of ation capable scheme the determine DIHl8.19 HCl R-DOI 12). (11, Turin by our described within first discussed as (36), methods work computational previous the used have 1B We Fig. Simi- can in exchanges discussion. deuterium found its of be with DAM-57, ease of for structure the highlighted larly, are exchanges terium 1 structure Fig. in as found to be the can (R)-DOI of on of pair effects structure a the for Chee calculated variants 5-HT (33), have deuterium well-characterized We several June of (35). and probability Oh tunneling Chee his- and for (34), by peak al. determined infrared et as characteristic a receptors with tidine agreement in also is cm 1,500–1,650 roughly the (36)—at pre- work above-discussed examining vious the by in ago- activation determined peak—as protein the active of assumed of theory den- vibrational efficacies probability this 5HT the of IET the with the at scaling determined; among nists behavior peak was displayed common agonists single sity the a of and tunnel- generated all study, were previous spectra this ing Within agonists. receptor serotonin auswr eemndb PDSP; by determined were Values structure of schemes deuteration Several 0 elto nteasmdatv ek uhadfeec hudbe should difference a Such peak. active assumed the in depletion ∼50% 6 DIHl8.02 HCl -DOI 2A hr (R)-DOI where , oeta h oain ftecniaedeu- candidate the of locations the that note 1; 2A (9.52) (6.45) 2A 3 PNAS eetr(6.Hri,w ettevalidity the test we Herein, (36). receptor n shratrrfre oa structure as to referred hereafter is and ± ± noag,add and orange, in K (K pKi n .78.65 0.07 .98.71 0.09 n (R and 6 = 2A | -O n A-7 The DAM-57. and (R)-DOI agonists: ) h5-HT i)* a 0 2017 30, May n )-d uldslcmn uvsexcept curves displacement full 2 = 6 -DOI 6 (2.24) (1.95) ngen oal,d Notably, green. in 2B ± ± | n K (K pKi .77.99 0.07 .77.96 0.07 7. = o.114 vol. stp xhneresults exchange Isotope ) eefe referred hereafter A, −1 1 eecniee to considered were 3 )h5-HT i) hseeg range energy this ; ,adte oh(d both then and ), | o 22 no. (10.23) (10.96) Alteration ) 6 2C ± ± eut in results K (K pKi 0.06 0.06 | 5597 2. 6 i) )

BIOPHYSICS AND COMPUTATIONAL BIOLOGY Table 2. Comparison of (R)-DOI and DAM-57 and their hexadeutero isotopologues for activation of 5-HT2A/2B/2C measuring Gq-mediated calcium flux, as illustrated in Fig. 4 Gq calcium flux

5-HT2A 5-HT2B 5-HT2C

EC50, nM Emax EC50, nM Emax EC50, nM Emax

Ligands (pEC50± SEM) % 5-HT (pEC50± SEM) % 5-HT (pEC50± SEM) % 5-HT 5-HT 0.83 100 1.29 100 0.25 100 (9.08 ± 0.02) (9.01 ± 0.12) (9.62 ± 0.10) (R)-DOI 0.58 95 ± 4 4.80 94 ± 1 2.19 101 ± 2 (9.25 ± 0.08) (8.43 ± 0.12) (8.70 ± 0.14)

(R)-d6-DOI 0.63 96 ± 4 4.40 91 ± 1 2.23 100 ± 3 (9.22 ± 0.10) (8.45 ± 0.10) (8.70 ± 0.15) DAM-57 1.54 98 ± 1 13.6 73 ± 2 57.5 81 ± 6 (8.82 ± 0.06) (7.87 ± 0.04) (7.24 ± 0.02)

d6-DAM-57 1.51 98 ± 1 12.6 74 ± 1 49.2 83 ± 6 (8.84 ± 0.07) (7.91 ± 0.05) (7.31 ± 0.02)

Calcium flux data were acquired with human 5-HT2A, 5-HT2B, and 5-HT2C INI-expressing tetracycline- inducible HEK cells. Estimates of EC50 and Emax represent the average and SE of the mean (SEM) from three independent experiments performed in triplicate. Emax is defined as percentage of 5-HT maximum response.

magnitude of an IET PDF peak associated with a mode is dependent on the Both (R)-DOI and (R)-d6-DOI were synthesized and were mass of an atom. This depletion of electron transfer should result in fewer assayed for binding affinity (Table 1). All four compounds were successful activations of the protein than the natural abundance compound. assayed for functional activity, using calcium flux assays (Fig. It is for these reasons that the deuteration schemes involving exchange of 4 and Table 2) at the 5-HT2A, 5-HT2B, and 5-HT2C receptors. all six of the methoxy hydrogens of DOI and the methyl chains on the amide Neither (R)-d6-DOI nor d6-DAM-57 presented significant alter- of DAM-57 were selected as candidates. ations in either binding or potency and efficacy (as noted by EC50 d6-(R)-DOI was prepared by an asymmetric synthesis, as shown in Fig. S1. NMR, mass spectral, and melting-point data were consistent with a pre- and Emax , respectively) compared with the protium versions. The viously published synthesis of the protio compound (87). d6-DAM-57 was endogenous agonist 5-HT was included in the calcium flux assays prepared by a standard route (Fig. S2), using either dimethylamine or d6- for comparison. dimethylamine. First, DOI and d6-DOI were submitted to the National Insti- The process of protein agonism/antagonism involves, at min- tute of Mental Health (NIMH)-sponsored Psychoactive Drug Screening Pro- imum, two steps: binding of the ligand to the active site of the gram (PDSP) (88) to determine their binding affinities at the human 5-HT2A protein and the activation of the protein. It should be noted that receptor and then all compounds were tested at 5-HT2A, 5-HT2B, and 5-HT2C these actions may happen in concert as proposed in the hand-in- receptors, measuring Gq-mediated calcium flux. A complete discussion of glove/multiconformation (5–8) models or as two individual steps the synthetic routes for all molecules, as well as a discussion of the biologi- cal assays, is given in Supporting Information. (11, 60, 67, 80, 90). In Table 1, we present the results of bind- ing displacement assays comparing the relative binding affinity Results and Discussion of both (R)-DOI and (R)-d6-DOI at several serotonin GPCRs. Herein, we operate under the following null hypothesis: A Alteration in the binding kinetics, as defined by pKi, shows no sig- vibration-sensitive mechanism is shared between all members nificant difference between the protonated and deuterated vari- of the GPCR class of proteins, possibly due to conserved topo- ants; explicitly, any difference found in the power-scaled equilib- graphical structures even without conservation of sequence iden- rium constant exists within the standard errors of the number, and tities, implying that general topographical structures of the therefore no claim of a difference can be made with confidence. GPCRs are essential in performing their biological task (89). Fol- The kinetic isotope effect may also affect the binding kinetics of lowing this logic, it seems reasonable that the family of GPCRs the G protein and thus could appear independently of ligand- likely shares a common fundamental activation mechanism. Fur- binding effects but would be apparent in activation studies. thermore, forms of both the lock-and-key and the glove-and- hand models exist in olfactory research, admitting acceptance of common aspects. Due to the likelihood of a common fun- damental activation mechanism, findings at CNS GPCRs will have implications at mammalian ORs. We have used a series of well-characterized GPCRs (explicitly h5-HT2A, h5-HT2B, and h5-HT2C) with two established ligands: (R)-DOI and DAM-57. IET spectra were generated for both molecules, using meth- ods prescribed by Turin in previous works (11, 12). Within a previous work (36), we examined the IET spectrum of several agonists and determined a consistently shared peak between the agonist’s spectra, the area of this shared spectral aspect roughly scaled with the agonist’s efficacy. This peak was consistent with the works of Chee and June (33), Chee et al. (34), and Oh (35), who attempted to generate a novel pharmacophore tool based on shared vibrational peaks. From the candidate deuter- Fig. 4. Calcium flux responses at human 5-HT2A, 5-HT2B, and 5-HT2C ation schemes discussed above, we sought the greatest possible INI receptors for (R)-DOI (red), (R)-d6-DOI (orange), DAM-57 (blue), and alteration in efficacy at the previously suggested activation peak d6-DAM-57 (purple). Data are representative at each receptor type per- to focus our experimental effort (36). formed in triplicate and in parallel.

5598 | www.pnas.org/cgi/doi/10.1073/pnas.1618422114 Hoehn et al. Downloaded by guest on September 28, 2021 Downloaded by guest on September 28, 2021 0 ag ,e l 21)Atredmninlmveo tutrlcagsi bacteri- in changes structural of movie three-dimensional A (2016) al. et E, Nango 10. 2 ui 20)Amto o h aclto foo hrce rmmlclrstruc- molecular from character odor of calculation the reception. for olfactory method A primary (2002) L for Turin mechanism 12. spectroscopic A (1996) L Turin 11. 3 afne ,Yyaa ,Fri 20)Ivsiaino irtoa hoyo olfac- of theory vibrational of Investigation (2001) J Fortin V, Yaylayan L, Haffenden 13. 5 aeS ta.(03 oeua irto-esn opnn nhmnolfaction. human in component vibration-sensing Molecular (2013) vibration-sensing al. et Molecular S, Gane (2011) 15. EMC Skoulakis A, Mershin L, Turin MI, Franco 14. 7 lc ,e l 21)Ipasblt ftevbainlter folfaction. of theory vibrational the of Implausibility (2015) al. et E, Block 17. smell the discriminate to learn mellifera) (Apis Honeybees (2014) al. et W, Gronenberg 16. ihnteCStruha E-su ehns,w tt that GPCRs state in we depen- mechanism, ligands the IET-esque of an evaluate through modes to CNS vibrational the was on within work activation this of protein— As dency effects. the pos- binding of of knowledge sible activation retaining directly—while the able VTO the are regarding addressing we conclusions aspects, draw both and considering to affinity com- By the activity. relevant both functional considered possibly the we with VTO, the conclude concerning and ments due exchange receptor to Kr isotope ligand Whereas of to paper binding recent (58). the in a receptor alterations of small histamine detected subject H2 the exam- were the been have and on protein 86) Isotope a (85, binding. with extensively ligand to a exchange, ined concern of isotopic this binding the of of on majority irrelevancy effects the the These relegate DAM-57. for do or argue but (R)-DOI not either do of findings activity the for exchange ligand– or found. ligand–receptor were effects either interactions) to protein ligand- receptor-G (due significant to activation no response or Therefore, the binding ligand). be of to 5-HT percentage taken endogenous the is the for 100% likewise (where parent–isotopologue and response SE of maximum the pair within each is between compounds, EC potency the in both ferences gives E which of 2, centage 5-HT Table all the in of at parent–isotopologue illustrated any of agonist pair at either full between compounds flux near It calcium in differ- species. a ence significant no five is 5-HT is prescient—there at for more DOI Additionally—and activity 2 agonist that Table partial 4 shows and 4 Fig. 5-HT Fig. from three both clear in is on found triplicate be dependent in can assay, performed was flux experiment (n calcium The by dissociation. Gαq determined expressing h5-HT was the cells vation of HEK members tetracycline-inducible all human at iments oh tal. et Hoehn .PneK ta.(06 etscn tutrldnmc rvstetascsisomeriza- trans/cis the drives dynamics structural Femtosecond (2016) al. et K, Pande into insights 9. Structural (2015) al. et of W, process Huang dynamic 8. The during (2013) changes al. conformational et R, of Nygaard Propagation 7. (2015) al. et R, of process Sounier dynamic the 6. into insights Structural (2015) al. et A, Manglik integrity membrane 5. of Role A (2011) receptors: K odorant Palczewski S, encode Filipek may A, family Debinski B, multigene Jastrzebska novel A 4. (1991) hip- R ventral Axel and reward. L, dorsal and cortex Buck the orbitofrontal of The 3. (2000) role ET The Rolls (2011) 2. W Ziegler MR, Hunsaker RP, Kesner 1. een efudn xetoa eednyo isotopic on dependency exceptional no found we Herein, exper- of series a through conducted was analysis Activation ini htatv elwprotein. yellow photoactive in tion 524:315–321. Cell activation. receptor signaling. receptor function. and stability Rhodopsin receptors: 50:267–277. protein-coupled G on recognition. odor for basis molecular memory. working olfactory in pocampus Senses orhodopsin. ture. inwt aiul aeldbenzaldehydes. labelled variously with tion cdSiUSA Sci Acad One 3802. in component fognccmonsfo hi epciedueae isotopomers. deuterated respective their 281:20133089. from compounds organic of flux Gαq the of Results statistics. relevant achieve to 3) = 152:532–542. 8:1–7. ho Biol Theor J 21:773–791. 2 Science 112:E2766–E2774. max eetr xmndi hsasy hra DAM-57 whereas assay, this in examined receptors rspiamelanogaster Drosophila 216:367–385. h oe-cldpEC power-scaled The . Cell Nature 354:1552–1557. 161:1101–1111. 524:375–378. 2 Science Cell eetrsbls.Rcpo acti- Receptor subclass. receptor olfaction. erbo er Mem Learn Neurobiol 2 65:175–187. 352:725–729. odChem Food eetr.Ti ocuinis conclusion This receptors. oii eetractivation. receptor µ-opioid 2B β rcNt cdSiUSA Sci Acad Natl Proc 2 arnri eetractivation. receptor -adrenergic ee cortex Cereb 50 n 5-HT and hc siae dif- estimates which , 73:67–72. 50 96:361–366. ˇ a ta.(58) al. et zan 10:284–294. 2C n h per- the and rgLpdRes Lipid Prog β receptors. 2 rcBo Sci Biol Proc -adrenergic 108:3797– rcNatl Proc µ-opioid Nature Chem PLoS 9 akrR,Bre L alrG 17)Temlclrbssfrsetdiscrimina- scent for basis molecular The (1973) GD Waller RL, Berdel RJ, evaluation in Barker Utilization pheromones: 29. Alarm (1971) M fly Beroza RE, melon Doolittle the MS, Blum of Deuteration 28. (1968) EL Schneider I, Keiser M, Beroza blending. RE, odour and Doolittle informa- vibration olfactory Molecular 27. (1983) of RH coding Wright Neural vibration: 26. molecular and Odor (1977) brain. R honeybee Wright the in isotopomers 25. of coding odour Differential (2016) al. et M, Paoli electro- 24. Differential (2016) EMC Skoulakis L, Turin K, Maniati A, Gaitanidis E, Drimyli 23. 3 heH,Jn S(03 oeua irto-ciiyrltosi nteaoimof agonism the in relationship vibration-activity Molecular (2013) OS June HK, Chee 33. Magalh ER, Maia perspective. 32. modern A relations: nitrobenzene-d Structure–odor to (2003) F Response Yoshii L, vibration: Turin molecular 31. and Odor (1975) RH Wright 30. 4,8-dimethyldecanal, of analogues Deuterated (2004) T Suzuki of S, spectra Matsuyama Raman J, II. Kim vibration. molecular 22. and Odour (1954) thermodynamic RSE and Serenius Quantum RH, I. Wright vibration. 21. molecular and Odour (1954) RH Wright 20. olfaction. of theory vibration the of test psychophysical A (2004) LB Vosshall A, Keller 18. 9 yo M(98 oeapcso h irto hoyo odor. of theory vibration the of aspects Some (1928) GM Dyson 19. upr rmteNtoa nttt fMna elhsosrdPsychoac- acknowledge Health-sponsored authors Mental Program. The of Screening Waybright. Drug Institute Jarod tive National by the analyses from MS support and NMR the ACKNOWLEDGMENTS. proteins. in within of ORs argue class of GPCR to exceptionalism the invoking difficult without VTO more the it of non-OR calls favor making within This additionally mechanism made IET while GPCRs. the GPCRs, predictions of nonolfactory viability herein. with within the question studied acting consistent into receptors VTO not 5-HT the are of under results series the our effect at Clearly, significant activity no has the iso- agonists hydrogen on DAM-57 the and of alteration (R)-DOI that at conclude or topes only potency can the we SE, in d the deviation for detected no was Similarly, efficacy flux. mea- be calcium function receptor to by in assumed changes sured be the were (R)-d cannot as therefore and significance, and any (R)-d (R)-DOI of SE both the between within binding for were the DOI potency in of deviations loss minor 50% of a d calculations as theory—predicted much Our Turin’s on as DAM-57. probability—based and tunneling the (R)-DOI CNS: family: the 5-HT the receptor in isotopologues—at specific expressed ligands—and widely characterized receptors serotonin h5-HT of contentious a series ORs—with of for a proposed viability activation—originally the protein examine of theory to attempted we Herein, Conclusion ORs—as general. both in GPCRs of (91)—and activation the for in suggested involved vibrational likely 5–8). than agree- refs. ligand—other quanta—are works: the in (recent of work theories properties our state Physiochemical transition places the this with Furthermore, ment strongly very VTO. argue paper the this against within theory. findings said the of that plausibility believe the We of suggestive evidence no found we 6 DM5,cmae ihterpoimcutrat.The counterparts. protium their with compared -DAM-57, in epnet nitrobenzene-d to Response tion: theories. olfactory of absorption. infra-red and response olfactory on Physiol effect Insect its and cue-lure, attractant, tion. Rep Sci antennae. drosophilid in isotopologues 3:015215. odorant to responses physiological nitrobenzene. of activity. pheromone aggregation the odour. nitrobenzene the with substances considerations. 19:456–459. Neurosci Nat dnsn receptors. adenosine odor. of understanding the towards Chem spectra Adv infrared molecules musk 457–515. for pp calculation Ed, 2nd Oxford), (CRC, RL Doty ed Gustation, and Olfaction nitrobenzene. with conditioned L.) mellifera (Apis bees honey ho Biol Theor J 2A 6:21893. aeldCmdRadiopharm Compd Labelled J h5-HT , 2014:1–13. 7:337–338. 14:1697–1712. Experientia plChem Appl J Drosophila e R,Lre A etoiuD ensa M(04 Quantum (2014) JM Bernassau D, Berthomieu DA, Lerner DRB, aes ˜ 64:473–502. 2B eoisInform Genomics netPhysiol Insect J n h5-HT and , h uhr rtflyakoldeassac with assistance acknowledge gratefully authors The PNAS 29:418–419. 4:611–615. | eetr ySbr n Seyed-allaei and Saberi by receptors 5 6 ytei n pheromonal and Synthesis castaneum: Tribolium DM5.A l auswr within were values all As -DAM-57. fhnybe ( bees honey of a 0 2017 30, May 17:2351–2361. 11:282–288. 47:921–934. plChem Appl J 2C ehv etdtowell- two tested have We . odtoe with conditioned L.) mellifera Apis | o.114 vol. 4:615–621. hmSenses Chem Experientia efmEsn i Rec Oil Essent Perfum | o 22 no. 6 DIand -DOI adokof Handbook 8:103–106. 31:530. | eNeuro 5599 5 6 of - J

BIOPHYSICS AND COMPUTATIONAL BIOLOGY 34. Chee HK, Yang JS, Joung JG, Zhang BT, Oh SJ (2015) Characteristic molecular vibra- 66. Moore CH, et al. (2012) Olfactory responses to explosives associated odorants are tions of adenosine receptor ligands. FEBS Lett 589:548–552. enhanced by zinc nanoparticles. Talanta 88:730–733. 35. Oh SJ (2012) Characteristics in molecular vibrational frequency patterns between ago- 67. Wang J, Luthey-Schulten ZA, Suslick KS (2003) Is the olfactory receptor a metallopro- nists and antagonists of histamine receptors. Genomics Inform 10:128–132. tein? Proc Natl Acad Sci USA 100:3035–3039. 36. Hoehn RD, Nichols D, Hartmut N, Sabre K (2015) Neuroreceptor activation by 68. Viswaprakash N, et al. (2009) Enhancement of odorant-induced responses in olfactory vibration-assisted tunneling. Sci Rep 5:9990. receptor neurons by zinc nanoparticles. Chem Senses 34:547–557. 37. Klika KD (2013) The potential of 13C isotopomers as a test for the vibrational theory 69. Vodyanoy V (2010) Zinc nanoparticles interact with olfactory receptor neurons. of olfactory sense recognition. ISRN Org Chem 2013:515810. BioMetals 23:1097–1103. 38. Turin L, Gane S, Georganakis D, Maniati K, Skoulakis EMC (2015) Plausibility of the 70. Hu C, Chan SI, Sawyer EB, Yu Y, Wang J (2014) Metalloprotein design using genetic vibrational theory of olfaction. Proc Natl Acad Sci USA 112:E3154. code expansion. Chem Soc Rev 43:6498–6510. 39. Block E, Jang S, Matsunami H, Batista VS, Zhuang H (2015) Reply to turin et al.: Vibra- 71. Seebungkert B, Lynch JW (2001) A common inhibitory binding site for zinc and odor- tional theory of olfaction is implausible. Proc Natl Acad Sci USA 112:E3155. ants at the voltage-gated K+ channel of rat olfactory receptor neurons. Eur J Neu- 40. Kaupp UB (2010) Olfactory signalling in vertebrates and insects: Differences and com- rosci 14:353–362. monalities. Nat Rev Neurosci 11:188–200. 72. Kinouchi K, Standifer KM, Pasternak GW (1990) Modulation of µ1, µ2, and δ opioid 41. Pellegrino M, Nakagawa T (2009) Smelling the difference: Controversial ideas in insect binding by divalent cations. Biochem Pharmacol 40:382–384. olfaction. J Exp Biol 212:1973–1979. 73. Standifer KM, Clark JA, Pasternak GW (1993) Modulation of µ1 opioid binding 42. Barwich AS (2016) What is so special about smell? Olfaction as a model system in by magnesium: Evidence for multiple receptor conformations. J Pharm Exp Ther neurobiology. Postgrad Med J 92:27–33. 266:106–113. 43. Crasto CJ (2009) Computational biology of olfactory receptors. Curr Bioinform 4:8–15. 74. Holst B, Elling CE, Schwartz TW (2002) Metal ion-mediated agonism and agonist 44. Abaffy T (2015) Human olfactory receptors expression and their role in non-olfactory enhancement in melanocortin MC1 and MC4 receptors. J Biol Chem 277:47662– tissues a mini-review. J Pharmacogenomics Pharmacoproteomics 6:152. 47670. 45. Kang N, Koo J (2012) Olfactory receptors in non-chemosensory tissues. BMB Rep 75. Li S, et al. (2016) Smelling sulfur: Copper and silver regulate the response of human 45:612–622. odorant receptor OR2T11 to low-molecular-weight thiols. J Am Chem Soc 138:13281– 46. Feldmesser E, et al. (2006) Widespread ectopic expression of olfactory receptor genes. 13288. BMC Genomics 7:121–121. 76. Block E (2017) Fifty years of smelling sulfur: From the chemistry of garlic to the molec- 47. Vanderhaeghen P, Schurmans S, Vassart G, Parmentier M (1993) Olfactory receptors ular basis for olfaction. Phosphorus Sulfur Silicon Relat Elem 192:141–144. are displayed on dog mature sperm cells. J Cell Biol 123:1441–1452. 77. Duan X, et al. (2012) Crucial role of copper in detection of metal-coordinating odor- 48. Flegel C, et al. (2016) Characterization of the olfactory receptors expressed in human ants. Proc Natl Acad Sci USA 109:3492–3497. spermatozoa. Front Mol Biosci 2:73. 78. Gerlach LO, et al. (2003) Metal ion enhanced binding of AMD3100 to Asp262 in the 49. Malki A, et al. (2015) Class I odorant receptors, TAS1R and TAS2R taste receptors, are CXCR4 receptor. Biochemistry 42:710–717. markers for subpopulations of circulating leukocytes. J Leukoc Biol 97:533–545. 79. Rodr´ıguez FI, et al. (1999) A copper cofactor for the ethylene receptor ETR1 from 50. Gaudin JC, Breuils L, Haertle´ T (2006) Mouse orthologs of human olfactory-like recep- Arabidopsis. Science 283:996–998. tors expressed in the tongue. Gene 381:42–48. 80. Solov’yov IA, Chang PY, Schulten K (2012) Vibrationally assisted electron transfer 51. Blache P, Gros L, Salazar G, Bataille D (1998) Cloning and tissue distribution of a new mechanism of olfaction: Myth or reality? Phys Chem Chem Phys 14:13861–13871. rat olfactory receptor-like (OL2). Biochem Biophys Res Commun 242:669–672. 81. Reese A, List NH, Kongsted J, Solov’yov IA (2016) How far does a receptor influence 52. Weber M, Pehl U, Breer H, Strotmann J (2002) Olfactory receptor expressed in ganglia vibrational properties of an odorant? PLoS One 11:1–21. of the autonomic nervous system. J Neurosci Res 68:176–184. 82. Checinska´ A, Pollock FA, Heaney L, Nazir A (2015) Dissipation enhanced vibrational 53. Pluznick JL, et al. (2009) Functional expression of the olfactory signaling system in the sensing in an olfactory molecular switch. J Chem Phys 142:025102. kidney. Proc Natl Acad Sci USA 106:2059–2064. 83. Tirandaz A, Taher Ghahramani F, Shafiee A (2015) Dissipative vibrational model for 54. Goto T, Salpekar A, Monk M (2001) Expression of a testis-specific member of the olfac- chiral recognition in olfaction. Phys Rev E 92:032724. tory receptor gene family in human primordial germ cells. Mol Hum Reprod 7:553– 84. Bittner ER, Madalan A, Czader A, Roman G (2012) Quantum origins of molecular 558. recognition and olfaction in Drosophila. J Chem Phys 137:22A551. 55. Drutel G, et al. (1995) Cloning of OL1, a putative olfactory receptor and its expression 85. Rakowski K, Paneth P (1996) Isotope effects on binding. J Mol Struct 378:35–43. in the developing rat heart. Recept Channels 3:33–40. 86. Swiderek´ K, Paneth P (2013) Binding isotope effects. Chem Rev 113:7851–7879. 56. Shulgin A, Shulgin A (1991) PIHKAL: A Chemical Love Story (Transform Press, Berkeley, 87. Mathis CA, Hoffman AJ, Nichols DE, Shulgin AT (1988) Synthesis of high specific activ- CA). ity 125I- and 123I-labelled enantiomers of 2,5-dimethoxy-4-iodophenylisopropylamine 57. Shulgin A, Shulgin A (1997) TIHKAL: The Continuation (Transform Press, Berkeley, CA). (DOI). J Labelled Comp Radiopharm 25:1255–1265. 58. Krzanˇ M, et al. (2016) The quantum nature of drug-receptor interactions: Deuteration 88. Besnard J, et al. (2012) Automated design of ligands to polypharmacological profiles. changes binding affinities for histamine receptor ligands. PLoS One 11:1–16. Nature 492:215–220. 59. Ushio-Fukai M (2009) Vascular signaling through G protein coupled receptors - new 89. Zhang Z, Wu J, Yu J, Xiao J (2012) A brief review on the evolution of GPCR: Conserva- concepts. Curr Opin Nephrol Hypertens 18:153–159. tion and diversification. Open J Genet 2:11–17. 60. Brookes JC, Hartoutsiou F, Horsfield AP, Stoneham AM (2007) Could humans recognize 90. Brookes JC, Horsfield AP, Stoneham AM (2012) The swipe card model of odorant odor by phonon assisted tunneling? Phys Rev Lett 98:038101. recognition. Sensors 12:15709–15749. 61. Lambe J, Jaklevic RC (1968) Molecular vibration spectra by inelastic electron tunnel- 91. Saberi M, Seyed-allaei H (2016) Odorant receptors of Drosophila are sensitive to the ing. Phys Rev 165:821–832. molecular volume of odorants. Sci Rep 6:25103. 62. Kirtley J, Scalapino DJ, Hansma PK (1976) Theory of vibrational mode intensities in 92. Curphey TJ (1979) Trifluoroacetylation of amino acids and peptides by ethyl trifluo- inelastic electron tunneling spectroscopy. Phys Rev B 14:3177–3184. roacetate. J Org Chem 44:2805–2807. 63. Phillips WA, Adkins CJ (1985) A theory for the intensities of inelastic electron- 93. Chambers JJ, Kurrasch-Orbaugh DM, Parker MA, Nichols DE (2001) Enantiospecific tunnelling spectra. Philos Mag B 52:739–750. synthesis and pharmacological evaluation of a series of super-potent, conformation- 64. Frazier JL, Heitz JR (1975) Inhibition of olfaction in the moth Heliothis virescens by ally restricted 5-HT2A/2C receptor agonists. J Med Chem 44:1003–1010. the sulfhydryl reagent fluorescein mercuric acetate. Chem Senses 1:271–281. 94. Stoll A, Hofmann A (1955) Amide der stereoisomeren lysergsauren¨ und dihydro- 65. Jia H, et al. (2016) Enhancement of odor-induced activity in the canine brain by zinc lysergsauren.¨ 38. Mitteilung uber¨ mutterkornalkaloide [Amides of the stereoisomers nanoparticles: A functional MRI study in fully unrestrained conscious dogs. Chem of lysergic acid and dihydrolysergic acid. 38. Report about Ergot alkaloids]. Helv Chim Senses 41:53–67. Acta 38:421–433. German.

5600 | www.pnas.org/cgi/doi/10.1073/pnas.1618422114 Hoehn et al. Downloaded by guest on September 28, 2021