...... HOT TOPICS 252

models with a tetrapeptide library Houghten RA, Dooley CT, Appel JR (2006). In vitro Unfortunately, the high degree of made up of a total of 6 250 000 peptides and direct in vivo testing of mixture-based combi- sequence homology between the 5- natorial libraries for the identification of highly active (200 mixtures made up of 125 000 and specific opiate ligands. AAPS J 8: E371–E382. HT2CR and 5-HT2BR has proved a tetrapeptides each) (Dooley et al, Houghten RA, Pinilla C, Giulianotti MA, Appel JR, major challenge in efforts to generate Dooley CT, Nefzi A et al (2008). Strategies for the 1998; Houghten et al, 2006, 2008). a truly specific high affinity 5-HT2CR use of mixture-based synthetic combinatorial Mixtures ranging from 2500 to libraries: scaffold ranking, direct testing in vivo, agonist that does not stimulate 5- 125 000 tetrapeptides have yielded and enhanced deconvolution by computational HT2BRs. Activity at the 5-HT2BRisa clear in vivo activity that is not methods. J Comb Chem 10: 3–19. particular concern because action at Liu B, Chang J, Gordon WP, Isbell J, Zhou Y, Tuntland necessarily related to classic in vitro T (2008). Snapshot pk: a rapid rodent in vivo these receptors is thought to contri- target-based screening. For mixture- preclinical screening approach. Drug Discov Today bute to the valvular heart disease based small molecule libraries the 13: 360–361. reported in some patients following process can be improved by careful Shukaliak Quandt J, Borras E, Prat E, Gelderblom H, fenfluramine-phentermine use (Fitz- Houghten RA, Kashani A et al (2004). Peptidic selection of those libraries guided by complex mixtures as therapeutic agents in cns gerald et al, 2000). Nevertheless, theoretical calculation of their drug- autoimmunity. Mol Immunol 40: 1075–1087. recent studies using combined phar- like properties. Over the past 10 years macological and genetic approaches in Neuropsychopharmacology Reviews (2009) 34, a process termed cassette testing (Liu 251–252; doi:10.1038/npp.2008.160 murine models have recently re- et al, 2008, and references cited there- kindled pharmaceutical interest in in) has been used to study in vivo drug discovery programs focusing on ADME with small mixture sets (typi- Targeting the serotonin generating more selective high affinity cally 5–10 related compounds) to 2C receptor for the 5-HT2CR agonists; for example, it has facilitate the early elimination of com- been shown that fenfluramine influ- pounds with poor drug-like profiling treatment of obesity and ences appetite through the melano- in PK profiling. type 2 diabetes cortin system (Heisler et al, 2002). The concept of using large, highly This brain pathway, specifically acting diverse mixture-based libraries for The neurotransmitter serotonin through the melanocortin 4 receptor, the identification of inherently more (5-hydroxytryptamine; 5-HT) has a is critical for the normal regulation of advanced ‘hits’ by the direct in vivo well-established role in energy homeo- energy balance, and integrates inputs testing is both exciting and promising. stasis. The clinical potential of bene- from many other neuropeptides and It remains to be seen if these ficially manipulating the 5-HT system neurotransmitters. recent early preliminary successes is best illustrated by the efficacy of More recently, a distinct role for the will fulfill their current potential compounds such as fenfluramine and 5-HT2CRs in glucose homeostasis has promise. sibutramine, both of which increase also been reported in rodents. Speci- F Ivy Carroll1 and 5-HT bioavailability, in the pharmaco- fically, both a classic 5-HT2CR agonist Richard A Houghten2,3 logical treatment of obesity. The (with binding affinity for other 5-HT 1Center for Organic and Medicinal Chemistry, Re- success of these compounds in redu- receptors) and a more selective and search Triangle Institute, Research Triangle Park, NC, cing food intake and body weight has high affinity 5-HT2CR agonist were USA; stimulated interest in dissecting the demonstrated to reduce elevated in- 2Torrey Pines Institute for Molecular Studies, San Diego, CA, USA; mechanisms whereby 5-HT influences sulin levels and improve glucose 3Torrey Pines Institute for Molecular Studies, Fort energy balance. Early pharmacological tolerance and insulin sensitivity in Pierce, FL, USA studies implicated the 5-HT2C receptor both genetically obese mice and in E-mail: [email protected] and [email protected] (5-HT2CR; previously classified as the mice with diet-induced obesity, both DISCLOSURE/CONFLICT OF INTEREST 5-HT1C receptor) in these effects, a with impaired glucose tolerance and The authors declare that except for income received suggestion supported by the later insulin resistance (Zhou et al, 2007). from their primary employers, no financial support or observation that genetic inactivation Importantly, these effects were compensation has been received from any individual or corporate entity over the past three years for of the 5-HT2CR, but not other 5-HT achieved at concentrations of the research or professional services, and there are no receptors, produces hyperphagia and compounds which were too low to personal financial holdings that could be perceived as obesity in the mouse (Tecott et al, influence food intake, energy expen- constituting a potential conflict of interest. 1995). 5-HT R knockout mice also diture, locomotor activity, or body ...... 2C Dooley CT, Ny P, Bidlack JM, Houghten RA (1998). display blunted responses to fenflur- weight. These findings indicate that Selective ligands for the m, d, and k opioid amine, indicating that action at these the 5-HT2CR may be a mechanistically receptors identified from a single mixture based receptors is required for the anorectic novel target for the treatment of type 2 tetrapeptide positional scanning combinatorial effect of this compound (Vickers et al, diabetes. This has been corroborated library. J Biol Chem 273: 18848–18856. Houghten RA (1994). Soluble combinatorial libraries: 1999). Collectively, these findings mo- by genetic inactivation of the 5-HT2CR extending the range and repetoire of chemical tivated attempts to develop selective in mice, which, either alone or in diversity. Methods Companion Methods Enzymol 5-HT2CR agonists for the treatment of combination with leptin deficiency, 6: 354–360. obesity. impairs glucose homeostasis (Wade

...... Neuropsychopharmacology HOT TOPICS ...... 253

et al, 2008). These findings identify a targets for drug discovery with approxi- relied on the parallel recording of up specific 5-HT receptor of relevance to mately 13% of marketed drugs having to 16 cells, in of itself a significant a prevalent metabolic disease. their mechanism of action attributed to increase in screening capability. A Together, these data indicate that activity at ligand- or voltage-gated ion recent innovation toward unprece- the 5-HT2CR is an attractive and channels (Overington et al, 2006). dented screening capacity has been tractable potential drug target for the Although this success is noteworthy, introduced in the form of a 48-channel treatment of obesity and/or type 2 there is a general consensus in the field QPatch system, a major advance over diabetes. Recent pharmaceutical ef- of ion channel-targeted drug discovery the manual recording approach where forts have led to the development of that progress has been significantly one can only imagine having 48 at least one compound that is cur- hampered because of the low-through- different individuals operating manual rently in clinical trials for obesity put nature of the gold standard assay recording set-ups. treatment. Results from these trials for electrophysiological assessment of Despite the obvious advantages as- are awaited with considerable ion channel activity, ie, manual patch sociated with fully automated ion interest. clamp electrophysiology in mammalian channel screening there are challenges Oliver J Marston1 and Lora K Heisler1 cell lines expressing channels of associated with the implementation of 1Department of Pharmacology, University of Cam- interest. Recent innovations in the these technologies. Not to be under- bridge, Cambridge, UK development of enabling technologies estimated is the often significant time E-mail: [email protected] supporting higher throughput and fully to generate a cell line compatible with ...... Fitzgerald LW, Born TC, Brown BS, Patterson JP, automated patch clamp electrophysiol- each platform, and not necessarily the Corjay MH, Valentine PA et al (2000). Possible role ogy (Dunlop et al, 2008; Lu and An, cell line you have been using for many of valvular serotonin 5-HT (2B) receptors in the 2008) have provided for a reenergizing years in others applications. This cardiopathy associated with fenfluramins. Mol of ion channel drug discovery with process together with assay optimiza- Pharmacol 57: 75–81. Heisler LK, Cowley MA, Tecott LH, Fan W, Low MJ, unprecedented capabilities for com- tion and validation can be lengthy and Smart JL et al (2002). Activation of central pound screening. resource intensive. However, these melanocortin pathways by fenfluramine. Science Two different approaches to achiev- challenges are clearly outweighed by 297: 609–611. Tecott LH, Sun LM, Akana SF, Strack AM, ing automation of manual patch the now unprecedented screening Lowenstein DH, Dallman MF et al (1995). Eating clamp electrophysiology have recently capability to support ion channel- 2C disorder and epilepsy in mice lacking 5-HT emerged taking advantage of the so- targeted drug discovery, holding much serotonin receptors. Nature 374: 542–546. Vickers SP, Clifton PG, Dourish CT, Tecott LH (1999). called planar array of multi-well con- promise for expediting the discovery Reduced satiating effect of d-fenfluramine in figurations in either a plate- or chip- of new ion channel-targeted drugs. serotonin 5-HT(2C) receptor mutant mice. Psycho- based format allowing for multiple pharmacology (Berl) 143: 309–314. John Dunlop1 Wade JM, Juneja P, MacKay AW, Graham J, Havel parallel recordings replacing the single 1 Neuroscience Discovery Research, Wyeth Re- PJ, Tecott LH et al (2008). Synergistic impairment channel recording typical of manual search, Princeton, NJ, USA of glucose homeostasis in ob/ob mice lacking patch clamp. The IonWorks platform E-mail: [email protected] functional serotonin 2C receptors. Endocrinology 149: 955–961. (Schroeder et al, 2003) was the first Zhou L, Sutton GM, Rochford JJ, Semple RK, Lam major innovation to be introduced DISCLOSURE/CONFLICT OF INTEREST DD, Oksanen LJ et al (2007). Serotonin 2C and although this technology did not The author is a full time employee of Wyeth Research receptor agonists improve type 2 diabetes via recapitulate the tight gigaohm seal ...... melanocortin-4 receptor signaling pathways. Cell Dunlop J, Bowlby M, Peri R, Vasilyev D, Arias R Metab 6: 398–405. quality typical of manual recordings, (2008). High-throughput electrophysiology: an a number of assays have been success- emerging paradigm for ion channel screening Neuropsychopharmacology Reviews (2009) 34, fully transferred onto this platform. and physiology. Nat Rev Drug Discov 7: 252–253; doi:10.1038/npp.2008.169 358–368. Most notably, it has been possible to John VH, Dale TJ, Hollands EC, Chen MX, Partington screen small compound libraries using L, Downie DL et al (2007). Novel 384-well the IonWorks (John et al, 2007), population patch clamp electrophysiology assays Turning up the pace of for Ca2+-Activated K+ channels. J Biomol Screen representing perhaps the best example ion channel screening in 12: 50–60. of how such technologies have revo- Lu Q, An WF (2008). Impact of novel screening drug discovery lutionized ion channel screening as technologies on ion channel drug discovery. Comb Chem High Throughput Screen 11: such a feat would be unimaginable 185–194. Ion channels represent an important with manual recording approaches. Overington JP, Al-Lazikani B, Hopkins AL (2006). family of integral membrane proteins The second series of technologies to How many drug targets are there? Nat Rev Drug Discov 5: 993–996. involved in many diverse physiological be introduced in the form of the Schroeder K, Neagle B, Trezise DJ, Worley J (2003). processes and are also implicated in a PatchXpress, QPatch, and Patchliner Ionworks HT: a new high-throughput electrophy- number of pathological conditions in (Dunlop et al, 2008) have successfully siology measurement platform. J Biomol Screen 8: 50–64. particular of the nervous, cardiovascu- recapitulated the gigaohm quality lar, and inflammatory systems. These seals typical of manual recordings. Neuropsychopharmacology Reviews (2009) 34, 253; proteins have proven to be attractive Until recently, these systems have doi:10.1038/npp.2008.163

...... Neuropsychopharmacology