Summary of probe development efforts to identify agonists of the transient receptor potential channel ML3 (TRPML3)
Name: Summary of probe development efforts to identify agonists of the transient receptor potential channel ML3 (TRPML3) ..more
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_AG_LEADS_SUMMARY
Name: Summary of probe development efforts to identify agonists of the transient receptor potential channel ML3 (TRPML3)
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. As a result, the identification of selective probes for TRPML3 would be useful to investigate the function of TRPML3 in inner ear mechanotransduction and hearing biology.
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.
Several important findings for these probes:
SID 24801657 (Powder SID 85786753)ML123:
(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)ML122:
(1) >20-fold selectivity over TPRN1 in Fluo-8 screening assay performed at Scripps.
(2) 60-fold selectivity over YFP-HEK parental cells in patch clamp assay performed by assay provider.
(3) 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://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html. A probe report for SIDs 24801657 and 24787221 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML123 and ML122, respectively. One manuscript has been published (14).
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 2010 Feb 2617(2):135-48.
15. Yamaguchi S, Muallem S. Opening the TRPML gates. Chem Biol. 2010 Mar 26;17(3):209-10.
Summary AID, summary, probes, TRPML3, 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.
Please see Related Bioassays and reference 14-15 for protocols performed in this probe development effort.
Categorized Comment - additional comments and annotations
Assay Cell Type: HEK293
From MLP Probe Report:
Probe count: 4
MLP Probe ML# for probe 1: ML123
PubChem Substance ID (SID) for probe 1: 24801657
PubChem Compound ID (CID) for probe 1: 2911646
Probe type for probe 1: Agonist
IC50/EC50 (nM) for probe 1: 873
Target for probe 1: TRPML3 (gi: 38174238)
Disease relevance for probe 1: Vertigo or tinnitus
Anti-target for probe 1: TRPN1
Fold selectivity for probe 1: 34
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK65432/ (ID: 2642658)
Grant number for probe 1: MH083077-01
MLP Probe ML# for probe 2: ML269
PubChem Substance ID (SID) for probe 2: 124349367
PubChem Compound ID (CID) for probe 2: 53239838
Target for probe 2: TRPML3 (gi: 38174238)
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK169454/ (ID: 3060534)
Grant number for probe 2: MH083077-01
MLP Probe ML# for probe 3: ML122
PubChem Substance ID (SID) for probe 3: 24787221
PubChem Compound ID (CID) for probe 3: 701237
Probe type for probe 3: Agonist
IC50/EC50 (nM) for probe 3: 1430
Target for probe 3: TRPML3 (gi: 38174238)
Disease relevance for probe 3: Vertigo or tinnitus
Anti-target for probe 3: TRPN1
Fold selectivity for probe 3: 20
MLP Probe ML# for probe 4: ML268
PubChem Substance ID (SID) for probe 4: 87692368
PubChem Compound ID (CID) for probe 4: 776924
PubMed Publication ID (PMID) for probe 1: 20189104
PubMed Publication ID (PMID) for probe 3: 20189104
NCBI Book chapter title for probe 1: Campaign to Identify Agonists of Transient Receptor Potential Channels 3 and 2 (TRPML3 & TRPML2)
NCBI Book chapter title for probe 2: Identification of Selective Agonists of the Transient Receptor Potential Channels 3 (TRPML3)
Data Table (Concise)