[01] Ruben M. Savizky, Department of Chemistry, The Cooper Union for the Advancement of Science and Art, New York, NY USA.

In this paper, a [2.2.1] bicyclic core is applied as a peptidomimetic scaffold capable of reproducing the structural relationship between the critical residues of the Tat protein, in an effort to create small molecules capable of binding to TAR RNA and inhibiting the Tat/TAR interaction. A systematic twenty-member panel mimicking an Arginine-Aspartic acid (R-D) moiety was synthesized and the ability of the individual members to inhibit the Tat/TAR interaction was evaluated. A gel-shift mobility assay was used to establish that several bicyclic agents inhibited the RNA-protein interaction with a micromolar level of activity.

HIV TAR RNA, RNA-small Molecule, Cycloaddition, Inhibitor

[01] Schroeder, R. and von Ahsen, U. (1996). Nucleic Acids and Molecular Biology (Eckstein, F. and Lilley, D. M. J., eds.), Springer-Verlag, Heidelberg, 53-74. [02] von Ahsen, U., Davies, J. and Schroeder, R. (1991). Antibiotic inhibition of group I ribozyme function. Nature 353:368-370. [03] Stage, T. K., Hertel, K. J. and Uhlenbeck, O. C. (1995). Inhibition of the hammerhead ribozyme by neomycin. RNA, 1: 95-101. [04] Rogers, J., Chang, A. H., von Ahsen, U., Schroeder, R. and Davies, J. (1996). Inhibition of the self-cleavage reaction of the human hepatitis delta virus ribozyme by antibiotics. J. Mol. Biol., 259: 916-925. [05] Draper, D. E. (1995). Protein-RNA recognition. Ann. Rev. Biochem., 64:593-620. Draper, D. E. (1999). Themes in RNA-protein recognition. J. Mol.. Biol., 293: 255-270. [06] Arnez, J. G. and Cavarelli, J. (1997). Structures of RNA-binding proteins. Q. Rev. Biophys., 30(3):195-240; Basu, S. and Bahadur, R. P. (2016) A structural perspective of RNA recognition by intrinsically disordered proteins. Cell Mol. Life Sci., Ahead of Print. [07] Ascano, M., Hafner, M., Cekan, P., Gerstberger, S. and Tuschl, T. (2012) Identification of RNA-protein interaction networks using PAR-CLIP. RNA, 3(2): 159-177; Re, A., Joshi, T., Kulberkyte, E., Morris, Q. and Workman, C. T. (2014). RNA-Protein Interactions: An Overview, Meth. Mol. Biol., 1097: 491-521. [08] Uhlenbeck, O. C., Pardi, A. and Feigon, J. (1997). RNA structure comes of age. Cell, 90:833-840; Marchaka, A. and Carlomagno, T. (2014). Solid-state NMR and RNA structure: A new partnership? eMagRes, 3(2):119-128 ; Fiscon, G., Paci, P. and Iannello, G. (2015), MONSTER v1. 1: a tool to extract and search for RNA non-branching structures, BMC Genomics, 16(Suppl.6):S1/1-S1/10; Achar, A. and Saetrom, P. (2015). RNA motif discover: a computational overview, Biol. Direct, 10: 61/1-61/22. [09] Grimm, D. (2009). Small silencing RNAs: State-of-the-art. Adv. Drug Del. Rev., 61(9):672-703; Millhavet, O., Gary, D. S., Mattson, M. P. (2003). RNA interference in biology and medicine. Pharm Rev. 55(4), 627-648; Hsu, P. D., Lander, E. S. and Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome editing. Cell, 157: 1262-1278. Abudayyeh, O. O., Gootenberg, J. S. Konermann, S., Joung, J., Slaymaker, I. M., Cox, D. B. T., Shmakov, S., Makarova, K. S., Semenova, E., Minakhin, L., Severinov, K., Regev, A. Lander, E. S., Koonin, E. V. and Zhang, F. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector, Science, 2016, first release June 2, 1-17. [10] Chow, C. S. and Bogdan, F. M. (1997). A Structural Basis for RNA-Ligand Interactions. Chem. Rev., 97:1489-1514. [11] Wilson, W. D. and Li, K.. (2001). Targeting RNA with small-molecules. Curr. Med. Chem., 7: 73-98. Philips, A., Milanowska, K., Lach, G. and Bujnicki, J. M. (2013) LigandRNA: computational predictor of RNA-ligand interactions, RNA, 19(12):1605-1616; Philips, A., Lach, G. and Bujnicki, J. M. (2015) Computational methods for prediction of RNA interaction with metal ions and small organic ligands. Meth. Enzym., 553: 261-285. [12] Xavier, K. A., Eder, P. S and Giordano, T. (2000). RNA as a drug target: methods for biophysical characterization and screening. Trends Biotech, 18:349-356. Da Veiga, C., Mezher, J., Dumas, P. and Ennifar, E. (2016) Isothermal Titration Calorimetry: Assisted Crystallization of RNA-Ligand Complexes, Meth. Mol. Biol., 1320, 127-143. [13] Gallego, J. and Varani, G. (2001). Targeting RNA with small-molecule drugs: Therapeutic promise and chemical challenges. Acc. Chem. Res., 34:836-843; Zacharias, M. (2003). Perspectives of drug design that targets RNA. Curr. Med. Chem.: Anti-Infective Agents, 2:161-172; Zaman, G. J. R., Michiels, P. J. A. and von Boeckel, C. A. A. (2003). Targeting RNA: new opportunities to address drugless targets. Drug Disc. Today, 8: 297-306. [14] Tran, T. (2014) The expansion and application of the RNA-small molecule partner database by two-dimensional combinatorial screening, in Stat Univ. of New York, Buffalo, NY, pp. 400; Velagapudi, S. P. (2014) Characterizing RNA-Small Molecule Interactions for the Design of Selective, Bioactive Small Molecules Targeting RNA from Sequence, in Stat Univ. of New York, Buffalo, NY, pp. 367. [15] Disney, M. D., Yildrin, I. and Childs-Disney J. L. (2014) Methods to enable the design of bioactive small molecules targeting RNA. Org. Biomol. Chem., 12(7): 1029-1039. [16] Hermann, T. and Westhof, E. (2000). Rational drug design and high-throughput techniques for RNA targets. Comb. Chem. & High Thr. Scr.., 3:219-234. Lorenz, D. A., Song, J. M. and Garner, A. L. (2015) High-throughput platform assay technology for the discovery of pre-microRNA-selective small molecule probes. Bioconj. Chem., 26(1): 19-23. [17] Hamy, F., Felder, E. R., Heizmann, G., Lazdins, J. and Aboul-ela, F. (1997). An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication. Proc. Natl. Acad. Sci. USA, 94: 3548-3553; Zeiger, M., Stark, S., Kalden, E., Ackermann, B., Ferner, J., Scheffer, U., Shoja-Bazargani, F., Erdel, V., Schwalbe, H. and Goebel, M. W. (2014) Fragment based search for small molecule inhibitors of HIV-1 Tat-TAR. Bioorg. Med. Chem. Lett., 24(24):5576-5580; Crawford, D. W., Blakeley, B. D., Chen, P.-H., Sherpa, C., Le Grice, S. F. J., Laird-Offringa, I. A and McNaughton, B. R. (2016) An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription. ACS Chem. Biol, Ahead of print. [18] Ludwig, V., Krebs, A., Stoll, M., Dietrich, U., Ferner, J., Schwalbe, H., Scheffer, U., Durner, G. and Gobel, M. W. (2007). Tripeptides from synthetic amino acids block the Tat-TAR association and slow down HIV spread in cell cultures. Chembiochem, 8(15):1850-1856; Mousseau, G., Mediouni, S. and Valente, S. T. (2015) Targeting HIV Transcription: the quest for a functional core. Curr. Topics Microbiol., Immunol., 389: 121-145. [19] Peytou, V., Condom, R., Patino, N., Guedj, R., Aubertin, A., Gelus, N., Bailly, C., Terreux, R and Cabral-Bass, D. (1999). Synthesis and Antiviral Activity of Ethidium-Arginine Conjugates Directed Against the TAR RNA of HIV-1. J. Med. Chem., 42:4042-4052; Wang, J., Wang, Y., Li, Z., Zhan, P., Bai, R., Pannecouque, C., Balzarini, J., De Clerq, E. and Liu, X. (2014) Design, Synthesis and Biological Evaluation of Substituted Guanidine Indole Derivatives as Potential Inhibitors of HIV-1 Tat-TAR Interaction. Med. Chem., 10(7): 738-746; Pascale, L., Gonzalez, A. L., Di Giorgio, A., Gayinski, M., Teixido Closa, J., Tejedor, R. E., Azoulay, S. and Patino, N. (2015) Deciphering structure-activity relationships in a series of Tat/TAR inhibitors. J. Biomol. Struct. Dyn., 1-54. [20] Litovchick, A., Evdokimov, A. G. and Lapidot, A. (1999). Arginine-aminoglycoside conjugates that bind to HIV transactivation responsive element RNA in vitro. FEBS Lett., 445:73-79; Joly, J.-P., Mata, G., Eldin, P., Briant, L., Fontaine-Vive, F., Duca, M. and Benhida, R. (2014) Artificial nucleobase-amino acid conjugates: a new class of TAR RNA binding agents, Chemistry, 20(7): 2071-2079. [21] Lapidot, A., Benasher, E. and Eisenstein, M (1995). Tetrahydropyrimidine Derivatives Inhibit Binding of a Tat-Like, Arginine-Containing Peptide, to HIV TAR RNA in vitro. FEBS Lett., 367: 33-38. [22] Hermann, T. and Westhof, E. (1998). RNA as a drug target: chemical, modelling, and evolutionary tools. Curr. Opin. Biotech., 9:66-73; Sukosd, Z., Andersen, E. S., Seemann, S. E., Jensen, M. K., Hansen, M., Gorodkin, J. and Kjems, J. (2015). Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain. Nuc. Acids Res., 43(21): 10168-10179. [23] Bayer, P., Kraft, M., Eichert, A., Westendorp, M., Frank, R. and Rösch, P. (1995). Structural studies of HIV-1 Tat protein. J. Mol. Biol., 247:529-535; Gu, J., Babayeva, N. D., Suwa, Y., Baranovskiy, A. GG., Price, D. H. and Tahirov, T. H. (2014) Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4. Cell Cycle, 13(11): 1788-1797. [24] Long, K. S. and Crothers, D. M.. (1995). Interaction of Human Immunodeficiency Virus Type 1 Tat-Derived Peptides with TAR RNA. Biochemistry, 34, 8885-8895. [25] Long, K. S. and Crothers, D. M.. (1999). Characterization of the Solution Conformations of Unbound and Tat Peptide-Bound Forms of HIV-1 TAR RNA. Biochemistry, 38, 10059-10069. [26] Tao, J. and Frankel, A. D. (1993). Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins. Proc. Natl. Acad. Sci. USA, 90: 1571-1575. [27] Puglisi, J. D., Tan, R. Y., Calnan, B. J., Frankel, A. D. and Williamson, J. R., (1992). Conformation of the TAR RNA-Arginine Complex by NMR Spectroscopy. Science, 257: 76-80. [28] Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., Karn, J., Lowe, A. D., Singh, M., Skinner, M. A. and Valerio, R. (1989). Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc. Natl. Acad. Sci. USA, 86: 6925-6929. [29] Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., Karn, J., Lowe, A. D., Singh, M. and Skinner, M. A. (1990). HIV-1 Tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure. EMBO J., 9(12): 4145-4153. [30] Churcher, M. J., Lamont, C., Hamy, F., Dingwall, C., Green, S. M., Lowe, A. D., Butler, J. G., Gait, M. J. and Karn, J. (1993). High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region. J. Mol.. Biol., 230: 90-110. [31] Weeks, K. M. and Crothers, D. M. (1991). RNA recognition by Tat-derived peptides: interaction in the major groove? Cell, 66:577-588. [32] Subramanian, T., Govindarajan, R. and Chinnadurai, G. (1991). Heterologous basic domain substitutions in the HIV-1 Tat protein reveal an arginine-rich motif required for transactivation. EMBO J., 10(8):2311-2318; Wu, C.-H., Chen, Y.-P., Liu, S.-L., Chien, F.-C., Mou, C.-Y. and Chen, R. P. (2015) Attenuating HIV Tat/TAR-mediated protein expression by exploring the side chain length of positively charged residues. Org. Biomol Chem., 13(45): 11096-11104. [33] Moras, D. and Poterszman, A. (1996). Protein-RNA interactions:getting into their major groove. Curr. Biol., 6(5): 530-532. [34] Aboul-ela, F., Karn, J. and Varani, G (1995). The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein. J. Mol. Biol., 253: 313-332. [35] Savinov, S. N. (2000). Design, synthesis and application of a hetero-bicycle-based peptidomimetic scaffold. In Yale Univ, New Haven, CT, USA, pp 305. [36] Park, W. K. C., Auer, M., Jaksche, H. and Wong, C. H. (1996). Rapid combinatorial synthesis of aminoglycoside antibiotic mimetics: Use of a polyethylene glycol-linked amine and a neamine-derived aldehyde in multiple component condensation as a strategy for the discovery of new inhibitors of the HIV RNA Rev responsive element. J. Am. Chem. Soc., 118:10150-10155; Ranjan, N., Kumar, S., Watkins, D., Wang, D., Appella, D. H. and Arya, D. P. (2013) Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates. Bioorg. Med. Chem. Lett., 23(20): 5689-5693. [37] Michael, K and Tor, Y. (1998). Designing novel RNA binders. Chem. Eur. J., 4:2091-2098. [38] Berlinck, R. G. S. (1996). Natural guanidine derivatives. Nat. Prod. Rep., 13:377-409; Berlinck, R. G. S. (1999). Natural guanidine derivatives. Nat. Prod. Rep., 16:339-365; Berlinck, R. G. S. (2002). Natural guanidine derivatives. Nat. Prod. Rep., 19:617-649; Berlinck, R. G. S. and Kossuga, M. H. (2005). Natural guanidine derivatives. Nat. Prod. Rep., 22:516-550; Berlinck, R. G. S. and Romminger, S. (2016) The chemistry and biology of guanidine natural products. Nat. Prod. Rep., 33(3): 456-490; [39] Alegre-Requena, J. V., Marques-Lopez, E. and Herrera, R. P. (2014) Guanidine motif in biologically active peptides. Australian J. Chem., 67(7):965-971; Sekutor, M. and Mlinaric-Majerski, K. (2015) Bioactive molecules – polycyclic guanidine derivatives. Kemija u Industriji, 64(3-4): 125-141. [40] Alonso-Moreno, C., Antinolo, A., Carrillo-Hemosilla, F. and Otero, A. (2014) Guanidines: from classical approaches to efficient catalytic syntheses. Chem. Soc. Rev., 43(10), 3406-3425; Tahir, S., Badshah, A. and Hussain, R. A. (2015) Guanidines from ‘toxic substance’ to compounds with multiple biological applications – Detailed outlook n synthetic procedures employed for the synthesis of guanidines. Bioorg. Chem., 59:39-79; Zhang, W.-X., De, S. and Chen, C. (2015) Syntheses of cyclic guanidine-containing natural products. Tetrahedron, 71(8):1145-1173; Lemrova, B. and Soural, M. (2015) Synthetic Strategies for Preparing Bicyclic Guanidines. Eur. J. Org. Chem., 2015(9): 1869-1886. [41] Savinov, S. N. and Austin, D. J. (1999). The diastereoselective cycloaddition of vinyl ethers with isomunchnones. Chem. Comm., 1813-1814. [42] Savizky, R. M. (2005). Synthetic and Biophysical Efforts Toward an Understanding of RNA Structure. In Yale Univ, New Haven, CT, USA, pp 263. [43] Johnson, E. C., Feher, V. A., Peng, J. W., Moore, J. M. and Williamson, J. R. (2003). Application of NMR SHAPES screening to an RNA target. J. Am. Chem. Soc., 125: 15724-15725. [44] Kotra, L. P., Haddad, J. and Mobashery, S. (2000). Aminoglycosides: Perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimic. Agents Chemo., 44: 3249-3256. [45] Swayze, E. E., Jefferson, E. A., Sannes-Lowery, K. A., Blyn, L. B. Risen, L. M., Arakawa, S., Osgood, S. A., Hofstadler, S. A. and Griffey, R. H. (2002). SAR by MS: A ligand based technique for drug lead discovery against structured RNA targets. J. Med. Chem., 45: 3816-3819. [46] Mei, H. Y., Mack, D. P., Galan, A. A., Halim, N. S., Heldsinger, A., Loo, J. A., Moreland, D. W., Sannes-Lowery, K. A., Sharmeen, L., Truong, H. N. and Czarnik, A. W. (1997). Discovery of selective, small-molecule inhibitors of RNA complexes. 1. The Tat protein TAR RNA complexes required for HIV-1 transcription. Bioorg. & Med. Chem., 5: 1173-1184. [47] Mischiati, C., Jeang, K. T., Feriotto, G., Breda, L., Borgatti, M., Bianchi, N. and Gambari, R. (2001). Aromatic polyamidines inhibiting the Tat-induced HIV-1 transcription recognize structured TAR-RNA. Antisense & Nuc. Acid Drug Dev., 11: 209-217. [48] Davis, B., Afshar, M., Varani, G., Murchie, A. I. H., Karn, J., Lentzen, G., Drysdale, M., Bower, J., Potter, A. J., Starkey, I. D., Swarbrick, T. and Aboul-ela, F. (2004). Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic “hot spots”. J. Mol. Biol., 336: 343-356. [49] Murchie, A. I. H., Davis, B., Isel, C., Afshar, M., Drysdale, M. J., Bower, J., Potter, A. J., Starkey, I. D., Swarbrick, T. M., Mirza, S., Prescott, C. D., Vaglio, P., Aboul-ela, F. and Karn, J. (2004). Structure-based drug design targeting an inactive RNA conformation: Exploiting the flexibility of HIV-1 TAR RNA. J. Mol. Biol., 336: 625-638. [50] Jefferson, E. A., Sprankle, K. G. and Swayze, E. E. (2000). Solid-phase synthesis of a heterocyclic ethylenediamine-derivatized library. J. Comb. Chem., 2: 441-444. [51] An, H. Y., Haly, B. D., Fraser, A. S., Guinosso, C. J. and Cook, P. D. (1997). Solution phase combinatorial chemistry. Synthesis of novel linear pyridinopolyamine libraries with potent antibacterial activity. J. Org. Chem., 62: 5156-5164. [52] Wang, Y., Hamasaki, K. and Rando, R. R. (1997). Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region. Biochemistry, 36: 768-779. [53] Huq, I., Wang, X. L. and Rana, T. M. (1997). Specific recognition of HIV-1 TAR RNA by a D-Tat peptide. Nat. Struct. Biol, 4: 881-882.