Supporting Information

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1 Supporting Information Superagonist, Full Agonist, Partial Agonist and Antagonist Actions of Arylguanidines at 5-Hydroxytryptamine-3 (5-HT 3 ) Subunit A Receptors Katie Alix, Shailesh Khatri, Philip D. Mosier, Samantha Casterlow, Dong Yan, Heather L. Nyce, Michael M. White, Marvin K. Schulte, and Małgorzata Dukat *, Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of Sciences, Philadelphia, PA 19104, USA Department of Biochemistry and Molecular Biology Drexel University College of Medicine, Philadelphia, PA 19102, USA *Corresponding author. Tel.: ; Fax: address: mdukat@vcu.edu (M. Dukat) Address: Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, PO Box , Richmond, VA , USA TABLE OF CONTENTS Content Table S1. C, H, N analysis for compounds 4 6. Figure S1. Dose response curves for agonists Figure S2. Inhibition of responses elicited by 2.5 µm serotonin for antagonists Figure S3. Sequence alignment of the mouse and human 5-HT 3A LBDs. Figure S4. Amino acid differences between the m5-ht 3 R and h5-ht 3A R. Figure S5. Modeled LBD C loop flexibility. Figure S6. Aryl guanidinium torsion angle preferences. Figure S7. Ligand binding mode identification workflow. Figure S8. Putative binding modes for agonists not shown in main text. Figure S9. Putative binding modes for antagonists not shown in main text. Figure S10. Relationships between affinity affinity (pk i ), potency (pec 50 /pic 50 ) and percent efficacy References Page S2 S3 S4 S5 S6 S7 S8 S9 S10 S12 S13 S15 S1

2 Table 1. C, H, N analysis for compounds 4 6. Calculated Found Compound Molecular Formula C H N C H N 4 C 7 H 8 FN 3 HNO C 7 H 8 BrN 3 HNO C 7 H 8 IN 3 HNO S2

3 Figure S1. Dose response curves for agonists The relative response (Y-axis) represents the current resulting from exposure of oocytes expressing m5-ht 3 A receptors to the test compound divided by the response obtained on the same oocyte from exposure to 2.5 µm serotonin (EC 90 ). S3

4 Figure S2. Inhibition of responses elicited by 2.5 µm serotonin for antagonists S4

5 Figure S3. Alignment of the primary amino acid sequences of the mouse and human 5-HT 3 A LBDs performed using CLUSTALX v.2 1 with default parameter settings. Residue numbers correspond to m5-ht 3 (top; UniProt ID: Q6J1J7; short splice variant) and h5-ht 3 A (bottom; UniProt ID: P46098). Secondary structural elements are depicted in gray, and the interfacial loops A G are indicated with colored bars. Residues whose side chains are part of the orthosteric binding site proximal to the aromatic box are indicated in cyan. Amino acid conservation between the mouse and human 5-HT 3 A sequences is colored according to the positively scoring groups in the Gonnet PAM250 matrix (identity = no color; strong residue similarity = green; weak similarity = yellow; no similarity = red). S5

6 Figure S4. Ribbon representation of two adjacent subunits of the homomeric 5-HT 3 receptor LBD from the mouse 5-HT 3 crystal structure (PDB ID: 4PIR). Differences in amino acid composition between the mouse 5- HT 3 and human 5-HT 3 A receptors are indicated by amino acid residues (ball-and-stick representation). The colors of the residues indicate the degree of conservation, as described in Figure S3. The colors of the principal and complementary loops also correspond to Figure S3. S6

7 Figure S5. LBD C loop flexibility as incorporated into the 5-HT 3 A models. Loop openness ranged from 9 to 25 Å between the C loop and back face centroids. The colors of the principal and complementary loops also correspond to Figure S3. The C loop conformation indicated in pink corresponds to a loop opening of 15 Å, from which the final docked solutions were derived. S7

8 Figure S6. Torsion angle preferences for the bond connecting the guanidinium group to the aromatic ring of mcpg (2). The mcpg (2) structure and torsion angle definition are shown, with the bond in question indicated in red. Computational systematic searches shown as potential energy curves identify low-energy conformational states for mcpg (2) (red line, Tripos Force Field; blue line, AM1). Experimentally-determined X-ray crystal structures for other arylguanidine-containing compounds are shown as dots (yellow, clonidine HCl crystal structure from Cody and DeTitta 2 ; blue, clonidine HCl crystal structure from Byre et al. 3 ; green, clonidine bound to bovine plasma copper-containing amine oxidase from Holt et al. [PDB ID: 2PNC] 4 ; red, 1-(mchlorophenyl)biguanide (mcpbg; 1) HCl crystal structure from López-Olvera and Soriano-García 5 ). S8

9 Figure S7. Ligand binding mode identification workflow. S9

10 Figure S8. Putative binding modes for agonists A) 3, B) 4, C) 5, D) 6, E) 7, F) 8 and G) 10. The receptor coloring scheme is the same as in Figure 3 (main text). A) 3 B) 4 C) 5 D) 6 E) 7 F) 8 S10

11 G) 10 S11

12 Figure S9. Putative binding modes for antagonists A) 12, B) 13, C) 14, D) 16 and E) 17. The receptor coloring scheme is the same as in Figure 3 (main text). A) 12 B) 13 C) 14 D) 16 E) 17 S12

13 Figure S10. A) Relationship between pk i and pec 50 or pic 50 for the arylguanidine compounds in this study. Filled circles = compounds with agonist activity; open circles = antagonists. B) Relationship between pk i and efficacy for the compounds with agonist activity. A) 10 compounds with agonist activity (filled circles, solid line): r = 0.91 antagonists (open circles, dashed line): r = pk i pec 50 (compounds with agonist activity) pic 50 (antagonists) S13

14 B) 10 r = pk i efficacy (%) S14

15 REFERENCES (1) Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J.;,Higgins, D. G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, (2) Cody, V., DeTitta, G. T. (1979) The molecular conformation of clonidine hydrochloride, an α- adrenergic agonist. J. Cryst. Mol. Struct. 9, (3) Byre, G., Mostad, A., Rømming, C. (1976) Crystal structure of clonidine hydrochloride, 2-(2,6- dichlorophenylamino)-2-imidazoline hydrochloride. Acta Chem. Scand. B 30, (4) Holt, A., Smith, D. J., Cendron, L., Zanotti, G., Rigo, A., Di Paolo, M. L. (2008) Multiple binding sites for substrates and modulators of semicarbazide-sensitive amine oxidases: kinetic consequences. Mol. Pharmacol. 73, (5) López-Olvera, G., Soriano-García, M. (2004) Crystal structure of 1-(m-chlorophenyl)biguanide hydrochloride. Anal. Sci. 20, x151 x152. S15