Neuron, Vol 36, 563-566, November 2002 Minireview Chemical Genetics: A Small Molecule Approach to Neurobiology Brian Koh1 and Craig M. Crews1,2,3 1 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520 USA 2 Department of Chemistry, Yale University, New Haven, CT 06520 USA 3 Department of Pharmacology, Yale University, New Haven, CT 06520 USA Correspondence: Craig M. Crews (203) 432-9364 (phone) (203) 432-5713 (fax) craig.crews@yale.edu Summary Chemical genetics, or the specific modulation of cellular systems by small molec ules, has complemented classical genetic analysis throughout the history of neur obiology. We outline several of its contributions to the understanding of ion ch annel biology, heat and cold signal transduction, sleep and diurnal rhythm regul ation, effects of immunophilin ligands, and cell surface oligosaccharides with r espect to neurobiology. Main Text Summary Main Text Selected Reading The contemporary neurological and psychiatric pharmacopoeia employs a staggering array of small molecules of both natural and synthetic origin to modulate patho logical processes. It is therefore only fitting that few areas of science have b enefited as much as neurobiology from the use of small molecules to explore cell ular processes. The specific and discrete perturbation of the cellular milieu by small molecules has been formalized as chemical genetics (Crews and Splittgerber, 1999 ; Mitchi son, 1994 ). The aims and strengths of this approach lie not in the recapitulati on of classical reverse genetics and traditional genetic manipulation. Rather, c hemical genetics complements genetic analysis by affording access to novel and p reviously hindered biological space. Among the foibles of classical genetics are limitations in the fine control of spatial and temporal dosimetry, as well as i ts awkwardness in dissecting cases of functional redundancy or the tangled skein s of complex signaling pathways. Ion Channel Biology and Small Molecules This strategy hardly constitutes a novel or uncommon approach in neurobiological research. One familiar example is MacKinnon's use of scorpion charybdotoxin as a specific and reversible inhibitor that enabled the mechanistic elucidation of cellular ionic currents in potassium channels. Using electrical methods, MacKinnon and colleagues demonstrated that scorpion to xin occluded potassium channels without altering their gating properties. Since this initial observation, scorpion toxin has been used liberally in the physical characterization of potassium channels. The Drosophila gene Shaker, the first p otassium channel to be cloned, was extensively mutagenized to identify the resid ues essential for toxin binding. These data provided not only a primary sequence potassium channel signature, but also clues to probable secondary and tertiary structural features. This in turn led to inferences regarding the physical size of the channel pore by careful analysis of the electrostatic interactions betwee n the toxin and the residues constituting the binding motif. Furthermore, the ef fort to crystallize the Streptomyces lividans potassium channel was validated by its interaction with scorpion toxin, suggesting that it was appropriately analo gous to its eukaryotic cousins at a deep level of evolutionary conservation (Hil le et al., 1999 ). With this precedent, neurobiologists have dissected the biology of ion channels by avidly exploiting the remarkable specificity of a diverse array of small mole cule toxins and venoms, many of which are considered the defining ligand for the ir respective targets (Bailey and Wilce, 2001 ; Lewis, 2000 ). Probing Heat and Cold Sensation with Small Molecules The power of a small molecule-based approach to receptor biology has been elegan tly illustrated in recent years through the identification of the receptors resp onsible for heat and cold sensation. It had been known for some time that capsai cin, the cardinal irritant and pungent component present in chili peppers (Figur e 1), was a highly selective compound unique among sensory neuron agonists as it elicited a refractory period of desensitization following the initial stimulato ry phase (Buck and Burks, 1986 ). In addition, Szallasi and Blumberg identified resiniferatoxin (RTX) as an especially potent but structurally divergent analog of capsaicin that shares a homovanillyl moiety critical for biological activity (Szallasi and Blumberg, 1990 ). The characterization of structure-activity relat ionships and the discovery of the capsaicin antagonist capsazepine (Walpole et a l., 1994 ) bolstered the notion of a vanilloid receptor, and its existence was f inally confirmed by autoradiographic visualization of a tritiated resiniferatoxi n probe in tissues of various species (Szallasi, 1995 ). Figure 1. Structures of Capsaicin, Resiniferatoxin, Capsazepine, and Menthol View larger version: [In this window] [In new window] Later, capsaicin was used as a molecular probe in the context of an expression c loning strategy to isolate the first nociceptive receptor, vanilloid receptor 1 (VR1). Characterization of VR1 revealed it to be a relative of the TRP ion chann el and a nonselective cation channel activated by capsaicin or elevated temperat ures (Caterina et al., 1997 ). Subsequently, other investigators identified the capsaicin-like substance N-arachidonoyl-dopamine (NADA) as a putative endogenous ligand (Huang et al., 2002 ). Intriguingly, a recent report identified another TRP-related ion channel based on its ability to transduce menthol or cold stimul ation. The discovery of this new receptor family member, CMR1 (cold- and menthol -sensitive receptor), suggests that TRP channels play a general role in the sign al transduction of thermal stimuli (McKemy et al., 2002 ). Sleep and Diurnal Rhythm Regulation by Small Molecules Chemical genetic approaches have proven equally facile with lipid-based compound s. For example, Cravatt and colleagues (1995 ) isolated the lipid oleamide, or c is-9,10-octadecenoamide (cOA) (Figure 2), from the cerebrospinal fluid of sleep- deprived cats, and demonstrated that exogenous synthetic oleamide induced sleep in rats. Figure 2. Structures of Diurnal Rhythm Regulators (Oleamide, Anandamide, 2-octyl -bromoace toacetate, and N-bromoacetyltryptamine) View larger version: [In this window] [In new window] Believing this to represent a novel signaling pathway, the investigators focused on elucidating an inactivating mechanism, and noted an enzymatic activity that hydrolyzed oleamide and the related fatty acid endocannabinoid anandamide into o leic and arachadonic acids, respectively. Mechanism-based isolation of this acti vity led to the purification and cloning of fatty acid amide hydrolase (FAAH) (C ravatt et al., 1996 ). This line of inquiry was supported by the observation tha t the human cerebrospinal fluid isolate 2-octyl -bromoacetoacetate, which had be en shown to lengthen REM-associated sleep in cats, also inhibited FAAH (Patricel li et al., 1998 ). Although oleamide and anandamide provoke ethological responses consistent with t he action of cannabinoids, only anandamide has been demonstrated to bind the can nabinoid receptor CB1. A recent study conducted with FAAH-deficient mice establi shed that both compounds are subject to catabolic regulation by FAAH, but that o nly anandamide's behavioral effects could be abolished by a deficiency of CB1 en gineered by either genetic means or pharmacological inactivation with the small molecule antagonist SR141716A (Lichtman et al., 2002 ). Hence, it appears that o leamide and anandamide exert their effects virtually orthogonally in vivo. Explo ration of the chemical space surrounding oleamide and anandamide has yielded a n umber of potent inhibitor analogs, which would be of utility in further characte rization of these effects (Boger et al., 2000 ). Various groups have shown interactions between oleamide and GABA(A) receptors, a nd ablation of the 3 subunit of GABA(A) receptors in a murine deficiency system abrogates oleamide's effects (Laposky et al., 2001 ). There are even suggestions that oleamide may possess additional properties, as it appears to interact with mammalian voltage-gated sodium channels in a manner reminiscent of many anaesth etics (Nicholson et al., 2001 ). In a related system, small molecules have been employed in the analysis of the m elatonin-pineal gland diurnal rhythm axis. The photoregulated enzyme serotonin N -acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]), transfers acety l from acetyl-coenzyme A (acetyl-CoASH) to serotonin in the first and rate-limit ing step of N-acetylserotonin formation en route to the biosynthesis of melatoni n (5-methoxy-N-acetyltryptamine). AANAT also possesses a secondary alkyltransfer ase activity, which is potently inhibited by the small molecule N-bromoacetyltry ptamine and its cognate N-haloacetyltryptamines. The acetyltransferase and alkyltransferase domains of AANAT are functionally dis tinct, and the mechanism of N-haloacetyltryptamine-mediated inactivation of AANA T is rather unusual. In brief, N-haloacetyltryptamines are prodrugs activated by transfer of CoASH at the alkyltransferase active site to generate species that subsequently bind and inhibit an AANAT acetyltransferase active site in either c is or trans. A more subtle consequence is the potential amplification of the inhibitory signa l, since the alkyltransferase site remains unaffected by the inhibition of the p hysiologically relevant acetyltransferase site. Hence, multiple N-haloacetyltryp tamine molecules can be converted to their inhibitory analogs even after their h ost enzyme's primary acetyltransferase activity has been neutralized. Cole and c olleagues have termed this type of inhibition ''molecular fratricide,'' and as t he N-haloacetyltryptamines are the only cell-permeable AANAT inhibitors describe d to date, these compounds hold much promise for the elucidation of melatonin's role in circadian rhythm biology (Zheng and Cole, 2002 ). Neurobiological Activities of Immunophilin Ligands Small molecules such as cyclosporin A (CsA), FK506, and rapamycin (Figure 3) are well characterized immunosuppressive compounds that form ligand-receptor comple xes with immunophilins, which in turn bind to and inhibit secondary targets such as calcineurin or FRAP/RAFT1 (FKBP and rapamycin-associated protein/rapamycin a nd FKBP12 target 1). These immunophilin ligands also exert a number of neurobiol ogically salient activities, including neuroprotective and neurotrophic effects for damaged neurons, modulation of neurotransmitter release secondary to NOS inh ibition, and nerve regeneration. Figure 3. Structures of Immunophilin Ligands (FK506, GPI-1046, and V-10,367) View larger version: [In this window] [In new window] In an effort to dissect the effects of calcineurin inhibition from these neurobi ological effects, rational drug design of a nonimmunosuppressive ligand, which b ound the immunophilin FKBP12, but not calcineurin, yielded 3-(3-pyridyl)-1-propy l (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate (GPI-1046). GPI-1046 acts as a neurotrophin that stimulates neurite outgrowth, and it has al so been credited with spurring axonal regeneration following serious CNS injury to either the optic nerve or spinal cord. It also exhibits neuroprotective effec ts of varying degrees against lesions induced by 3-acetylpyridine, hydrogen pero xide, 6-hydroxydopamine, 1-methyl-4-phenylpyridinium, and parachloroamphetamine. There is some evidence for a protective elevation of antioxidative glutathione levels, although strong evidence for any direct target is lacking. Interestingly , insult by 1-methyl-4-phenylpyridinium to both wild-type neuronal cells treated with GPI-1046 and their FKBP12-deficient counterparts treated with FK506 result in similar neuroprotective profiles, which further supports a distinction betwe en the immunosuppressive and neuroprotective effects of immunophilin ligands. Another nonimmunosuppressive immunophilin ligand analog, V-10,367, also spurs ne urite outgrowth in dopaminergic neuronal cell culture in addition to the acceler ation of nerve regeneration in a rat model and neuroprotection in a murine traum atic brain injury (TBI) model. One group used the novel compound Lie120, a highl y specific calcineurin inhibitor, to distinguish the neuroprotective effects of FK506 and V-10,367 from calcineurin inhibition. Their results suggested that cal cineurin inhibition was strictly orthogonal to the neuroprotective effects of bo th FK506 and V-10,367 in cell culture (Snyder et al., 1998a, 1998b ) . Modulation of Cell Surface Chemistry Using Small Molecules Finally, we wish to address a model study of the specific perturbation of a cell ular system with small molecules to generate novel insights into neuronal biolog y that complements genetic analysis. Linear homopolymers of the saccharide 2,8-s ialic acid (poly-2,8-sialic acid [PSA]), are primarily concentrated on the cell surface upon neural cell adhesion molecules (NCAM) and play an important role in neuronal development and synaptic plasticity. Manipulation of PSA levels was pr eviously limited to either enzymatic digestion or genetic means. The latter appr oach was inherently problematic, as multiple distinct enzymatic steps are respon sible for the synthesis of oligosaccharides such as endogenous PSA. Bertozzi and colleagues (Mahal et al., 2001 ) recently described the modulation of this oligosaccharide epitope by using the small molecule N-butanoylmannosamin e (ManBut) (Figure 4) to inhibit PSA generation in a specific and reversible man ner. ManBut is permitted in lieu of the natural substrate N-acetylmannosamine (M anNAc) in the cellular biosynthetic pathway leading to cytidine 5'-monophosphate -sialic acid (CMP-sialic acid), the quantum of PSA fabrication, and is metabolic ally transformed into an unnatural chain-terminating derivative when incorporate d into 2,8-sialic acid oligomers undergoing extension. The closely related compo und N-propanoylmannosamine (ManProp) is also a suitable substrate for incorporat ion into unnatural sialic acid derivative, but does not interfere with the itera tive catenation that leads to PSA and its analogs, rendering it an excellent mat ched control. Figure 4. Structures of poly-2,8-sialic acid (PSA) Precursors (N-acetylmannosamine [ManNAc ], N-propanoylmannosamine [ManProp], and N-butanoylmannosamine [ManBut]) View larger version: [In this window] [In new window] In this system, tunable inhibition and temporal potentiation of PSA expression i s but a matter of adulterating endogenous sialic precursor pools with ManBut in a dose-dependent fashion. ManBut treatment at extremely low doses would alter th e nature of population of PSA to include both full-length as well as truncated P SA molecules. Alternatively, increasing amounts of ManBut would attenuate both t he population and mean length of PSA molecules by prematurely terminating PSA ol igomers on average at increasingly earlier stages. This chemical genetic approac h will enable finer resolution of PSA epitope function in neuronal processes thr ough tractable methods (Mahal et al., 2001 ). Conclusion We have attempted to illustrate how chemical genetic analysis of various neurobi ological processes offers additional analytical power in juxtaposition to tradit ional forward and reverse genetic analysis. The chemical genetic approach is lim ited theoretically only by the level of specificity and bioavailability of the s mall molecules marshaled in its employ. That said, precise target identification and validation remain significant challenges in the elucidation of chemical gen etic pathways. As evidenced by the continuous struggle to recapitulate the synthesis of natural products, the exploitation of chemical space tendered by small molecule-based s caffolds is not yet a mature science. The promise of this genetic paradigm is ne cessarily tempered by our collective chemical prowess and creativity. Acknowledgments C.M.C. gratefully acknowledges support from the National Institutes of Health (G M62120). Selected Reading Summary Main Text Selected Reading Bailey P. and Wilce J. (2001) Venom as a source of useful biologically active mo lecules. Emerg. Med., 13:28-36. Boger D.L., Sato H., Lerner A.E., Hedrick M.P., Fecik R.A., Miyauchi H., Wilkie G.D., Austin B.J., Patricelli M.P. and Cravatt B.F. (2000) Exceptionally potent inhibitors of fatty acid amide hydrolase: the enzyme responsible for degradation of endogenous oleamide and anandamide. Proc. Natl. Acad. Sci. USA, 97:5044-5049 . [Medline] Buck S.H. and Burks T.F. (1986) The neuropharmacology of capsaicin: review of so me recent observations. Pharmacol. Rev., 38:179-226. [Medline] Caterina M.J., Schumacher M.A., Tominaga M., Rosen T.A., Levine J.D. and Julius D. (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathw ay. Nature, 389:816-824. [Medline] Cravatt B.F., Prospero-Garcia O., Siuzdak G., Gilula N.B., Henriksen S.J., Boger D.L. and Lerner R.A. (1995) Chemical characterization of a family of brain lipi ds that induce sleep. Science, 268:1506-1509. [Medline] Cravatt B.F., Giang D.K., Mayfield S.P., Boger D.L., Lerner R.A. and Gilula N.B. (1996) Molecular characterization of an enzyme that degrades neuromodulatory fa tty-acid amides. Nature, 384:83-87. [Medline] Crews C.M. and Splittgerber U. (1999) Chemical genetics: exploring and controlli ng cellular processes with chemical probes. Trends Biochem. Sci., 24:317-320. [M edline] Hille B., Armstrong C.M. and MacKinnon R. (1999) Ion channels: from idea to real ity. Nat. Med., 5:1105-1109. [Medline] Huang S.M., Bisogno T., Trevisani M., Al-Hayani A., De Petrocellis L., Fezza F., Tognetto M., Petros T.J., Krey J.F. and Chu C.J. et al. (2002) An endogenous ca psaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proc. Natl. Acad. Sci. USA, 99:8400-8405. [Medline] Laposky A.D., Homanics G.E., Basile A. and Mendelson W.B. (2001) Deletion of the GABA(A) receptor beta 3 subunit eliminates the hypnotic actions of oleamide in mice. Neuroreport, 12:4143-4147. [Medline] Lewis R.J. (2000) Ion channel toxins and therapeutics: from cone snail venoms to ciguatera. Ther. Drug Monit., 22:61-64. [Medline] Lichtman A.H., Hawkins E.G., Griffin G. and Cravatt B.F. (2002) Pharmacological activity of fatty acid amides is regulated, but not mediated, by fatty acid amid e hydrolase in vivo. J. Pharmacol. Exp. Ther., 302:73-79. [Medline] Mahal L.K., Charter N.W., Angata K., Fukuda M., Koshland D.E. Jr. and Bertozzi C .R. (2001) A small-molecule modulator of poly-alpha 2,8-sialic acid expression o n cultured neurons and tumor cells. Science, 294:380-381. [Medline] McKemy D.D., Neuhausser W.M. and Julius D. (2002) Identification of a cold recep tor reveals a general role for TRP channels in thermosensation. Nature, 416:52-5 8. [Medline] Mitchison T.J. (1994) Towards a pharmacological genetics. Chem. Biol., 1:3-6. 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(1990) Resiniferatoxin and its analogs provide nov el insights into the pharmacology of the vanilloid (capsaicin) receptor. Life Sc i., 47:1399-1408. [Medline] Walpole C.S., Bevan S., Bovermann G., Boelsterli J.J., Breckenridge R., Davies J .W., Hughes G.A., James I., Oberer L. and Winter J. et al. (1994) The discovery of capsazepine, the first competitive antagonist of the sensory neuron excitants capsaicin and resiniferatoxin. J. Med. Chem., 37:1942-1954. [Medline] Zheng W. and Cole P.A. (2002) Serotonin N-acetyltransferase: mechanism and -- ※ 来源:．生命玄机站 bbs.cst.sh.cn. [FROM: WWW]
