Poster C1-609 Novartis Institutes for BioMedical Research, Inc. Omadacycline (PTK796) Mechanism of Action Studies by Using In Vitro Protein Synthesis 4560 Horton Street Session 87 Emeryville, CA 94608 Phone: 510-923-2057, Fax: 510-923-5550 Inhibition Assay and Molecular Modeling Email: [email protected] A. RUZIN, S. MULLIN, P. PETRONE, L. WHITEHEAD and P. A. BRADFORD − Novartis Institutes for BioMedical Research, Cambridge, MA

Figure 1. Structures of molecules used in this study. Computational Chemistry ••In comparison with tetracycline (Figure 3A), additional C. Omadacycline predicted binding pose using Cresset B. Omadacycline interaction map. ABSTRACT favorable molecular interactions are made in the C1054/G1053 alignment to tetracycline. N N N N The Thermus thermophilus X-ray solution of tetracycline within ADE966 GUA 966 H2N H H H H H H HO HO the 30S subunit (accession code 1HNW) was used for generating binding area with the additional tertiary-amine-containing N N O O O O CYT1054 H P O HO H H P N Background H H CYT1195 OH H N N H N side-chains of both omadacycline and tygecycline. H OHOH O 2 2 N the modeling hypotheses. The ConQuest small molecule X-ray H N N O N N 2 Omadacylcine (OMC) is a novel aminomethylcycline anti- OH OH H URA1196 OH O O O OH O OH O O OH O OH database for ligand three dimensional atomic structures was also ••Omadacycline has a secondary amine functionality capable of O O OH O OH N N bacterial compound. The structural similarity between OMC and H H H O used in the preparation of ligand structures. Docking calculations HO URA interacting with the phosphate backbone oxygens on G1053 O 531 tetracyclines (known inhibitors of bacterial protein synthesis) Omadacycline (PTK796) Tigecycline CYT HO 1054 P O of the ligands was performed in the GLIDE docking suite H2N via a salt-bridge of distance 3.4Å and a small hydrophobic O O such as tigecycline (TGC), minocycline (MINO) and tetracycline (Schrodinger Inc.) and molecular alignments generated with OH H interaction with C1054 within 2.4Å (Figures 3C and 4B). CYT1195 OH O O OHOH O OHOH N O N N (TET) as well as previous studies suggested that OMC targets N N N H HO H H H H H Cresset (Cresset BMD Ltd.). Visualisation of these results was O 2+2+ HO HO O bacterial protein synthesis. This study assessed the potency of MgM OH ••Tigecycline is also capable of interacting via a 5.2Å salt bridge O O H performed in the Maestro (Schrodinger Inc.) and ICM (Molsoft) O H N H N HO OMC as compared to TGC, MINO and TET by using an in vitro 2 2 5.4 Å P with the phosphate of G1053 and 6.8Å coulombic interaction 2.4Å O OH OH applications. O O transcription/translation (IVT) inhibition assay. Computational O O OH O OH O O OH O OH P GUA1053 with the phosphate on U531, as well as the amide moiety O O 2+ OH OH Mg chemistry studies by structure based design and molecular Minocycline Tetracycline π-stacking with C1054 within 2.7Å (Figures 3B and 4A). O O O HO P O N modeling using alignment and docking techniques provided RESULTS Mg2+ HO insight into ligand binding. ••An alternate binding pose was also postulated by the in-silico 3.4Å N H Previous studies showed that tigecycline specifically inhibits ••The results of the IVT assay are shown in Figure 2 and Table 1. calculations, tigecycline and omadacycline binding within a URA O protein synthesis in the in vitro coupled transcription-translation GUA1053 1052 The IC50 values for tigecycline, omadacycline, minocycline Methods (IVT) assay with potency 3- and 20-fold greater than that of cleft coordinating two magnesium ions in the groove formed and tetracyline were 0.9, 2.8, 3.5 and 13.8 µM, respectively. minocycline and tetracycline, respectively.3 The aim of this by G963, A964, U1052, G1053, G1197, G1198 & U1199 IVT reactions were conducted by using an E. coli S30 extract Percent inhibition values at 10 µM of tigecycline, omadacycline, system and pBESTluc plasmid DNA as a template. Luciferase study was to assess the potency of omadacycline in the IVT assay (Figure 3D). D. An alternate binding mode for tigecycline and omadacycline minocycline and tetracyline were 99.4, 78.7, 77.6 and 49.9%, CONCLUSIONS activity was assayed on LmaxII luminometer. Data analysis as compared to tigecycline, minocycline and tetracycline. predicted with GLIDE. Coordination of two magnesium cations respectively. Figure 3. Tetracycline pose in the X-ray structure and was performed using Microsoft Office Excel 2007 and Grafit by carbonyl oxygens and hydroxyl functionalities on each ring ••The results of this study confirmed that the mechansim of action The X-ray crystal structure of tetracycline (Protein Data Bank 5 (v5.0.4). GLIDE docking and Cresset alignment molecular molecular docking of tigecycline and omadacycline. of the tetracyclic chemical scaffold. of omadacycline is the inhibition of protein synthesis. code 1HNW) within T. thermophilus 30S ribosome was solved Figure 2. Effect of tetracycline, minocycline, tigecycline modeling technologies were used to predict binding poses using The nucleotide residues are pointed with the white arrows. The ••From comparison of IC50 and % percent inhibition values, by Brodersen et al.4 The proximity of the ligand in relation to and omadacycline on the protein synthesis in the IVT a Thermus thermophilus X-ray solution, pdb code 1HNW. distances between the atoms are shown with the yellow lines. the results of this study suggest that omadacycline is a more nucleotides C1195, U1196, C1054 and G1053 and a magnesium assay. URA1196 potent in vitro protein synthesis inhibitor than minocycline and Results cation was used as the basis of modeling experiments utilising Data analysis was performed using Grafit 5 (v5.0.4; Erithacus A. Tetracycline pose in the 1HNW X-ray structure. pharmacophore alignment and molecular docking experiments tetracycline while less efficient inhibitor than tigecycline. Software Ltd.). RLU – relative luminescense units. N HO IC50 values for TGC, OMC, MINO and TET in the IVT assay H H of tigecycline and omadacycline. HO CYT1195 ••Based on the molecular modeling, several factors may be at 60000 60000 H CYT1054 were 0.9, 2.8, 3.5 and 13.8 µM, respectively. Percent inhibition H2N OH URA play for tigecycline as compared to omadacycline: values at 10 µM of TGC, OMC, MINO and TET were 99.4, 78.7, O O OH O OH 1196 40000 40000 ––the larger surface area of a π interaction between the amide U 77.6 and 49.9%, respectively. Modeling suggested that secondary MATERIALS AND METHODS U CYT1195 RL RL CYT moiety of tigecycline with the pyrimidine of C1054 amine group of OMC may form a salt bridge with the phosphate 1054 Mg2+ In vitro assay for inhibition of coupled in vitro transcription- 20000 20000 2.4Å on guanine 1053 and a weak hydrophobic interaction with ––the amine salt bridge to guanine phosphate groups is shared translation (IVT). The IVT assay utilized E. coli S30 extract 5.4Å 3.4Å by both molecules and is likely equivalent in strength cytosine 1054. In comparison, TGC may form a salt bridge 0 0 system for circular DNA (Promega, Madison, WI). Plasmid 10-1 1 101 10-1 1 101 with the phosphate on G1053, a coulombic interaction with the Mg2+ 4.8Å ––closer inter-atomic proximity of the salt bridge to U531 for pBESTluc™ was used as a template encoding luciferase. All Tetracycline Minocycline Parameter Value Std. Error Parameter Value Std. Error 2.5Å phosphate on U531 and an amide moiety π-stacking with C1054. Mg2+ 4.7Å GUA1053 the amine in tigecycline compounds were solubilized from powders in nuclease-free H2O Y Range 67071.9646 4934.0998 Y Range 51040.0427 3686.1909 An alternate pose was also found for both TGC and OMC binding IC 50 13.8142 2.2645 IC 50 3.5284 0.5541 2.7Å ––one fewer rotatable bond in the side-chain results in lower to 40 X final assay concentration. The assay was performed as Slope factor 1.0028 0.1101 Slope factor 1.6580 0.3631 within a cleft coordinating two Mg++ ions in the groove formed Background -11902.5690 4478.4095 Background 1486.4076 2759.9803 5.4Å URA1050 rotational entropy compared to omadacycline that has four follows: 60000 60000 by G963, A964, U1052, G1053, G1197, G1198 and U1199. Mg2+ GUA1053 rotatable bonds

••Each sample was tested in duplicates. 40000 40000 Figure 4. Interaction maps. ––tigecycline’s amide N-H potentially being locked in an U Conclusions U ••Pipetted 3.5 µl of 286 ng/µl pBESTluc™ into the wells RL RL Charge interactions are shown with the red arrows, H-bonding intramolecular hydrogen bond with the phenolic hydroxyl. The results of this sudy confirmed that the mechansim of action 20000 20000 with the green arrows, hydrophobic contacts with the blue arrows. of 384-well assay plate. Negative control wells received H2O of OMC is the inhibition of protein synthesis. The data suggested B. Tigecycline predicted binding pose using Cresset alignment only. A. Tigecycline interaction map. REFERENCES that OMC was a more efficient inhibitor than MINO and TET 0 0 to tetracycline. 10-2 10-1 1 101 10-1 1 101 1. Macone, A., J. Donatelli, T. Dumont, S. B. Levy, and S. K. Tanaka. 2003. Presented at the Transferred 0.5 µl of 40 X compound to assay wells. Positive Tigecycline Omadacycline rd although it is a less efficient inhibitor than TGC. Molecular •• N N 43 Interscience Conference on Antimicrobial Agents and Chemotherapy, abstract F-754. H ADE GUA H2N Parameter Value Std. Error Parameter Value Std. Error HO H H 966 966 O 2. Weir, S., A. Macone, J. Donatelli, C. Trieber, D. E. Taylor, S. K. Tanaka, and S. B. Levy. 2003. modeling data are consistent with the results of IVT assay. control wells (no compound) received 0.5 µl of H2O. Y Range 51763.7265 1702.2066 Y Range 56370.8025 2156.0303 H O O O O CYT1054 H N N P IC 50 0.9386 0.0704 IC 50 2.7929 0.2418 2 N P N Presented at the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy, abstract OH OHOH OH Slope factor 1.6448 0.1792 Slope factor 1.2905 0.1362 O O OH O OH H O O F-751. ••Pipetted 16 µl of master mix (contains 2 µl of amino acid Background -677.7686 1114.8547 Background -789.2316 1513.9617 N O 3. Olson, M. W., A. Ruzin, E. Feyfant, T. S. Rush, 3rd, J. O’Connell, and P. A. Bradford. 2006. mix, 8 µl of S30 premix and 6 µl of S30 extract) into assay URA1196 N N INTRODUCTION H H H O Functional, biophysical, and structural bases for antibacterial activity of tigecycline. Antimicrob HO URA Agents Chemother 50:2156-66. wells. Table 1. Comparison of the IC50 and percent inhibition CYT1054 O O 531 HO P O 4. Brodersen, D. E., W. M. Clemons, Jr., A. P. Carter, R. J. Morgan-Warren, B. T. Wimberly, and V. Omadacycline (7-dimethylamino, 9-(2,2-dimethyl-propyl)- H2N values from the IVT assay N O O Ramakrishnan. 2000. The structural basis for the action of the antibiotics tetracycline, pactamycin, ••Incubated plate for 1 hr at 37°C. OH aminomethylcycline) is a novel antibacterial agent that is CYT OH O N N and hygromycin B on the 30S ribosomal subunit. Cell 103:1143-54. URA 1195 O O OHOH O OHOH H HN H 1 531 2+2+ active against both Gram-positive and Gram-negative bacteria. ••Added an equal volume (20 µl) of room temperature Compound IC50 values, μM % inhibition at 10 μM 2.7Å O O MgM OH ® O O Omadacycline is structurally similar to protein synthesis inhibitors Steady-Glo Luciferase assay reagent (Promega, Madison, WI) HO O 5.4 Å P 2.5Å O from the tetracycline family (Figure 1). In vitro macromolecular tetracycline 13.8 49.9 O O to all assay wells. 2+ 6.8Å Mg P GUA1053 synthesis assays with radiolabeled substrates have demonstrated O O 2+ OH OH Mg ••Incubated 20 minutes at room temperature and read minocycline 3.5 77.6 O O O that omadacycline inhibited protein synthesis while having no 5.2Å HO P O N luminescence with the luminometer. 5.4Å GUA1053 significant effect on RNA, DNA and peptidoglycan synthesis and tigecycline 0.9 99.4 HO N H 2+ was able to compete for tetracycline binding sites on bacterial Data analysis was performed using Microsoft Office Excel 2007 Mg O Poster presented at 51st Interscience Conference on Antimicrobial Agents and Chemotherapy. 2 omadacycline 2.8 78.7 URA1052 ribosomes. and Grafit 5 (v5.0.4; Erithacus Software Ltd.). Sept. 17-20. Chicago