Late stage results from the probe development efforts to identify agonists of the Transient Receptor Potential Channels 3 and 2 (TRPML3 and TRPML2).
Name: Late stage results from the probe development efforts to identify agonists of the Transient Receptor Potential Channels 3 and 2 (TRPML3 and TRPML2). ..more
BioActive Compounds: 6
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Stefan Heller, Stanford University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH083077-01
Grant Proposal PI: Stefan Heller, Stanford University
External Assay ID: TRPML3-TRPML2_AG_PROBES_LATE STAGE
Name: Late stage results from the probe development efforts to identify agonists of the Transient Receptor Potential Channels 3 and 2 (TRPML3 and TRPML2).
Cell signaling pathways that mediate osmosensation, photosensation, and thermosensation depend on a family of diverse transient receptor potential (TRP) cation channels, which are activated by agonist-receptor coupling (1-5). A role for these channels in inner ear hair cell mechanotransduction was gleaned from TRP channel mutations identified in flies, worms, and lower vertebrates with defective balance and impaired sensitivity to touch (1-5). TRPML3 (mucolipin 3; MCOLN3) is a TRP channel expressed in inner ear hair cells and stereocilia (5-7), suggesting it may play a role in hearing and mechanotransduction. Reports that mice with mutations in TRPML3 (known as varitint-waddler mutants) exhibit early-onset hearing loss accompanied by head-bobbing and circling behaviors (8-10), provided further support for a role of TRPML3 in hearing and vestibular function. TRPML2 is a human cation channel, previously known as Mucolipin 2, is encoded by the MCOLN2 gene (11). TRPML2 is associated with the Arf6-regulated trafficking pathway and is involved in the intracellular transport of membranes and membrane proteins (12), including TRPML3 (13). As a result, the identification of probes for TRPML3 and TRPML2 would be useful to investigate the function of TRPML3 in inner ear mechanotransduction and hearing biology, as well as elucidate pathways of intracellular transport of membrane proteins. There are currently no agonists for TRPML2 or TRPML3.
Summary of Probe Development Effort:
Following primary HTS in singlicate to identify TRPML agonists (AID 1448), confirmation of hit activity in triplicate (AID 1526), counterscreening against TRPN1 (AID 1525), titration assays to determine compound potency (AID 1562) and selectivity (AID 1682), compounds belonging to secondary arylsulfonamide (SIDs 24801657, 14746905, 3716245, and 17414375) and sulfonarylpiperazine scaffolds (SIDs 24787221 and 24792359) were identified as possible candidates for probe development. The chemical series selected for optimization had the potential to meet the criteria for a successful probe as laid out in the CPDP, and in all cases to improve the state of the art in the field.
The above probe candidates were reordered as powders (SIDs 85786753, 85786755, 85786754, 85786756; SIDs 85786752, and 85786751) and tested by the SRIMSC or assay provider in the following assays: TRPML3 dose response assay (Assay 1), TRPN1 dose response assay (Assay 2), TRPML3 patch clamp (Assay 3), patch clamp counterscreening on parental YFP-HEK cells (Assay 4), and Fura-2 imaging assays against TRPML3 (Assay 5) and TRPML2 (Assay 6). The most potent compound from each scaffold (SID 85786753 and SID 85786752) was identified as a probe, with EC50 values of 0.45 micromolar and 2.0 micromolar, respectively. Results of the Fura-2 assays revealed that these probes have activity against both TRPML3 and TRPML2. Importantly, these assays also revealed that these probes are inactive against hTRPML1, hTRPM2, mTRPV2, hTRPC3, drTRPN1, and hTRPA1 ion channels.
Several important findings for these probes:
SID 24801657 (Powder SID 85786753)
(1) >34-fold selectivity over TPRN1 in Fluo-8 screening assay performed at Scripps.
(2) 183-fold selectivity over YFP-HEK parental cells in Patch clamp assays performed by assay provider.
(3) Activates TRPML3 & TRPML2 in Fura-2 profiling assays performed by assay provider.
SID 24787221 (Powder SID 85786752)
a. >20-fold selectivity over TPRN1 in Fluo-8 screening assay performed at Scripps.
b. 60-fold selectivity over YFP-HEK parental cells in patch clamp assay performed by assay provider.
c. Activates TRPML3 & TRPML2 in Fura-2 profiling assays performed by assay provider.
These compounds are an improvement over the prior art. The results of these probe development efforts resulted in the identification of two TRPML3/TRPML2 agonist probes belonging to unique chemical scaffolds.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below. The results of our probe development efforts can be found at http://molscreen.florida.scripps.edu/probes.shtml. One manuscript has been published (14). Please also see Summary AID 1809.
1. Clapham, D.E., TRP channels as cellular sensors. Nature. 2003. 426(6966): p. 517-24.
2. Cuajungco, M.P., C. Grimm, and S. Heller, TRP channels as candidates for hearing and balance abnormalities in vertebrates. Biochim Biophys Acta. 2007. 1772(8): p. 1022-7.
3. Gillespie, P.G. and R.G. Walker. Molecular basis of mechanosensory transduction. Nature. 2001. 413(6852): p. 194-202.
4. Eberl, D.F., R.W. Hardy, and M.J. Kernan. Genetically similar transduction mechanisms for touch and hearing in Drosophila. J Neurosci. 2000. 20(16): p. 5981-8.
5. Qian F, Noben-Trauth K. Cellular and molecular function of mucolipins (TRPML) and polycystin 2 (TRPP2). Pflugers Arch. 2005 Oct;451(1):277-85.
6. Atiba-Davies M, Noben-Trauth K. TRPML3 and hearing loss in the varitint-waddler mouse. Biochim Biophys Acta. 2007 Aug;1772(8):1028-31.
7. Gong, Z., W. Son, Y.D. Chung, J. Kim, D.W. Shin, C.A. McClung, Y. Lee, H.W. Lee, D.J. Chang, B.K. Kaang, H. Cho, U. Oh, J. Hirsh, M.J. Kernan, and C. Kim. Two interdependent TRPV channel subunits, inactive and Nanchung, mediate hearing in Drosophila. J Neurosci. 2004. 24(41): p. 9059-66.
8. Di Palma, F.; Belyantseva, I. A.; Kim, H. J.; Vogt, T. F.; Kachar, B.; Noben-Trauth, K. Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice. Proc. Nat. Acad. Sci. 99: 14994-14999, 2002.
9. Nagata K, Zheng L, Madathany T, Castiglioni AJ, Bartles JR, Garcia-Anoveros J. The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration. Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):353-8.
10. van Aken AF, Atiba-Davies M, Marcotti W, Goodyear RJ, Bryant JE, Richardson GP, Noben-Trauth K, Kros CJ. J Physiol. 2008 Sep 18. TRPML3 mutations cause impaired mechano-electrical transduction and depolarization by an inward-rectifier cation current in auditory hair cells of varitint-waddler mice.
11. Clapham DE, Julius D, Montell C, Schultz G (December 2005). "International Union of Pharmacology. XLIX. Nomenclature and structure-function relationships of transient receptor potential channels". Pharmacol. Rev. 57 (4): 427-50. doi:10.1124/pr.57.4.6.
12. Karacsonyi C, Miguel AS, Puertollano R (October 2007). Mucolipin-2 localizes to the Arf6-associated pathway and regulates recycling of GPI-APs. Traffic 8 (10): 1404-14. doi:10.1111/j.1600-0854.2007.00619.
13. Venkatachalam K, Hofmann T, Montell C. Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1. J Biol Chem. 2006 Jun 23;281(25):17517-27.
14. Small molecule activators reveal that TRPML3 integrates multiple stimulation modes. Grimm C, Jors S, Saldanha SA, Obukhov AG, Pan B, Oshima K, Cuajungco MP, Chase P, Hodder P, and Heller S. Chemistry & Biology (in press).
Late stage, probes, nonselective, TRPML3, TRPML2, mucolipin 2, TRP cation channel, mucolipin 3, MCOLN3, deafness, HEK 293, agonist, agonism, activator, activation, fluorescence, calcium, Fluo-8 dye, dose response, counterscreen, 1536, HTS, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
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Please see AIDs 1448, 1525, 1526, 1562, 1682, and Summary AID 1809 for protocols performed in this probe development effort.
Additional probe development assays are described below.
TRPML3 Agonist Assay (Assay 1):
The purpose of this assay is to determine dose response curves for test compounds that act as agonists of the TRPML3 cation channel. This assay employs a HEK293 cell line that stably expresses the human TRPML3-YFP cation channel. The cells are treated with test compounds followed by measurement of intracellular calcium as monitored by a fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as TRPML3 agonists will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye, and thus increase well fluorescence. Compounds are tested in triplicate in a 10-point 1:3 dilution series starting at a nominal concentration at a concentration 29.9 micromolar.
TRPN1 Agonist Counterscreen Assay (Assay 2):
The purpose of this assay is to determine dose response curves for test compounds that act as agonists of the TRPN1 cation channel. This assay also serves as counterscreens for the TRPML3 assays. This assay employs a HEK293 cell line that stably expresses the zebrafish TRPN1-YFP cation channel. The cells are treated with test compounds followed by measurement of intracellular calcium as monitored by a fluorescent, cell permeable calcium indicator dye. As designed, compounds that act as TRPN1 agonists will increase calcium mobilization, resulting in increased relative fluorescence of the indicator dye, and thus increase well fluorescence. Compounds are tested in triplicate in a 10-point 1:3 dilution series starting at a nominal concentration at a concentration 29.9 micromolar.
Patch Clamp Assays (Assays 3 and 4):
The purpose of these assays is to determine if test compounds can increase current recordings in TRPML3 ion channels (Assay 3) or the YFP-HEK parental background (Assay 4). Whole-cell currents were recorded with an Alembic Instruments VE-2 amplifier with 100% series resistance compensation, and acquired with JClamp software. The standard bath solution contained (in mM) 138 NaCl, 5.4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 d-glucose, adjusted to pH 7.4 with NaOH. The standard pipette solution contained (in mM) 140 CsCl, 10 HEPES, 3 ATP-Na, 1 BAPTA, and 2 MgCl2, adjusted to pH 7.2. 100 uM 2-Aminoethyl-diphenyl borate was included in the bath solution to block gap junctions and had no effect on the expressed channels. Channel responses were plotted to 10 ms voltage steps (holding potential = +10 mV) between -200 mV and +100 mV in 20 mV incremental steps, normalized by cell capacitance (pF). Compounds were tested at 10 micromolar.
Fura-2 Assays (Assays 5 and 6):
The purpose of these assays is to determine whether compounds identified as probe candidates are able to increase whole cell Ca2+ influx in HEK293 cells transfected with human TRPML3, other human, or murine (m) TRP channels, or zebrafish TRPN1. In this assay cells transiently expressing channels or YFP control plasmid are perfused with test compound, followed by measurement of intracellular [Ca2+] for 2 minutes with the fluorescent indicator fura-2-AM. Compounds are added to cells 20-25 hours after transfection. Values are reported as mean values +/- SEM (n >= 3 independent experiments with 20-30 cells). The % activation values for TRPML2 in the SAR tables were calculated by normalizing the TRPML2 response ratios to TRPML3 response ratios. Compounds are tested at 10 micromolar.
In this assay the PubChem Activity Score is assigned a value of 100 for probe compounds, 50 for analogs and 0 for inactives.
Probes were identified. Analog SID 85786751 was not tested in patch clamp and Fura-2 assays because of its lower potency against TRPML3, compared to the probes.
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