Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4860–4864 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT Frank Sielaff a, Eva Böttcher-Friebertshäuser b, Daniela Meyer a, Sebastian M. Saupe a, Ines M. Volk a, Wolfgang Garten b, Torsten Steinmetzer a,⇑ a b Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, D-35032 Marburg, Germany Institute of Virology, Philipps University Marburg, Hans-Meerwein-Str. 2, D-35043 Marburg, Germany a r t i c l e i n f o Article history: Received 19 May 2011 Revised 8 June 2011 Accepted 9 June 2011 Available online 21 June 2011 Keywords: HAT Protease inhibitor Serine protease Influenza Hemagglutinin cleavage a b s t r a c t A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the Dconfiguration as P3 residue. Whereas inhibitor 4 (Ki 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, Ki 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HATexpressing MDCK cell model. Ó 2011 Elsevier Ltd. All rights reserved. Human influenza viruses cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks and occasional pandemics worldwide.1 Currently, two drugs targeting the viral neuraminidase (NA) and the M2 channel blockers amantadine and rimantadine are approved for the treatment of influenza. Resistance to these drugs have been observed and exacerbate the situation.2,3 The replication cycle of influenza viruses is initiated by its surface glycoprotein hemagglutinin (HA). HA mediates the binding of the virus to sialic acid containing receptors of the host cells and, after endocytosis, the fusion of the viral envelope with the endosome membrane. This process is termed uncoating and enables the release of the viral genomic RNA into the cytosol of the host cell.4 HA is synthesized as HA0 precursor and has to be cleaved by host endoproteases into disulfide-linked HA1 and HA2 subunits to become fusogenic and is thus a crucial step for infectivity and spread of influenza viruses. The HAs of most influenza strains, including the H1, H2 and H3 subtypes, which typically infects humans, contain a single arginine as the P1 residue at its cleavage site.5 Recently it was shown that the trypsin-like serine proteases HAT (human airway trypsin-like protease or TMPRSS11D), TMPRSS2 (epitheliasin or transmembrane protease serine 2) and TMPRSS4 (CAP 2 or transmembrane protease serine 4), which are expressed in the human respiratory tract, efficiently cleave the HA0 of various influenza strains with a monobasic cleavage site.6,7 ⇑ Corresponding author. Tel.: +49 6421 2825900; fax: +49 6421 2825901. E-mail address: steinmetzer@staff.uni-marburg.de (T. Steinmetzer). 0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2011.06.033 In addition, HAT and TMPRSS2 also support the multicycle influenza virus replication in HAT- or TMPRSS2-expressing MDCK cells.6 Additionally, we could demonstrate that HAT is proteolytically active on the cell surface, whereas it seems that TMPRSS2 cleaves HA0 in the secretory pathway within the cell.8 Due to its location on the cell surface and its better accessibility HAT might be a potential target for the treatment of influenza infections. Recently, a first peptidic arginal-derived HAT inhibitor with a Ki value of 54 nM has been developed.9 HAT belongs to the type II transmembrane serine proteases and has a mosaic-like structure with its protease domain in the C-terminal part.10 So far no X-ray structure of HAT is available. However, the sequence of its protease domain has significant similarity to other trypsin-like serine proteases, such as the clotting proteases thrombin11 and factor Xa12 or the fibrinolytic urokinase (uPA).13 These proteases are all inhibited by substrate analogue structures containing a 4-amidinobenzylamide as a decarboxylated P1 arginine mimetic in combination with a P3 amino acid in the D-configuration.14,15 Starting from a homology model16–18 of HAT based on the X-ray structure of DESC119 (2oq5.pdb) we assumed that such 4-amidinobenzylamide derivatives should be suitable HAT inhibitors. A first screen with available compounds, like the previously described factor Xa inhibitor 112 and the uPA inhibitors 2 and 313 revealed also some inhibition of HAT with Ki-values >50 nM. These analogues have been previously used for preliminary experiments to demonstrate the inhibition of proteolytic activation and propagation of influenza viruses in HAT-expressing MDCK cells.20 4861 F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4860–4864 P3-P2 NH S O O P3 Ki (nM) 1 D-Arg-Gly 430 2 D-Ser-Ser 78 3 D-Ser-Lys(Cbz) 51 21 NH2 H N P2 Therefore, we screened22 and synthesized additional analogues of this inhibitor type. Based on previous experience with related thrombin,23 factor Xa12 and urokinase13 inhibitors it was known that proline is a suitable P2 residue in substrate analogue inhibitors of various trypsin-like serine proteases. Consequently, we prepared a series of P2 proline analogues with several P3 amino acids in the D-configuration and maintained the important P4 benzylsulfonyl15 and the P1 4-amidinobenzylamide24 (Table 1). Only the replacement of glycine in inhibitor 1 by proline resulted in an approximately 20-fold improved inhibition constant of compound 5, which could marginally be enhanced by incorporation of a P3 homo-D-arginine (4, Ki 13 nM). Obviously, the conformationally constrained proline is well accepted by the S2 site of HAT. However, proline is also a suitable P2 residue for many other trypsin-like serine proteases and its incorporation often leads to lack of specificity. Indeed, compound 5 is also a potent inhibitor of thrombin (3.5 nM), factor Xa (2.4 nM), plasma kallikrein (8.3 nM), matriptase (55 nM) and matriptase 2 (170 nM), thus completely lacking any selectivity.25,26 The other P2 proline analogues have reduced potency, however, some of them still inhibit HAT with Ki-values <100 nM, including several inhibitors with hydrophobic P3 residues. Based on the Ki-values it seems that HAT has some preference for positively charged P3 residues, whereas the acidic D-aspartyl residue (17) is poorly accepted. Interestingly, the tert-butyl protected D-aspartic- or D-glutamic acid inhibitors 6 and 7 show relatively high potency with Ki-values <50 nM. Therefore, we used analogues with a free D-aspartic- and D-glutamic acid side chain in the P3 position as suitable starting point for further modifications and incorporated various piperazine derivatives and other cyclic amines in an additional series (Table 2). The strongest inhibitory potency within this series was obtained with a 1-(2-pyrimidyl)-piperazine coupled to the side chain of D-glutamic acid (18). However, all other analogues had reduced activity; so we decided to keep D-arginine in the P3 position to study the influence of further P2 substitutions. Results from Table 3 indicate that valine, isoleucine and alanine as well as its close analogues a-aminobutyric acid and norvaline are suitable P2 residues and provide inhibitors with Ki-values 6 30 nM. We assumed that some of these analogues could have an improved selectivity compared to the unspecific proline derivative 5. Therefore, we selected some inhibitors and determined their Ki-values against the coagulation proteases thrombin and factor Xa and the fibrinolysis enzyme plasmin (Table 4). Inhibitor 31 is the only compound that has stronger potency against HAT compared to the other proteases. Especially compounds 4 and 5 have a significantly higher affinity to thrombin and factor Xa. The inhibitory potency against thrombin could be strongly reduced by incorporation of a P2 serine residue (36). A similar effect was previously observed in a series of analogous urokinase inhibitors13; however, inhibitor 36 is still a relatively potent factor Xa inhibitor. In contrast, all selected compounds from the piperazine series (18, 19, 21) showed a reduced affinity for factor Xa but were still relatively potent thrombin inhibitors (Ki <15 nM). It should be noted that various compounds also inhibit the protease domain of TMPRSS2 with Ki-values <100 nM (e.g., 20, 53 and 68 nM for derivatives 5, 30 and 31, respectively). Detailed results regarding the Table 2 Inhibition of HAT by inhibitors of the general structure NH O H N O S O H N NH2 N O n O Compound n 18 2 Ki (nM)21 P3 N N N 17 N O 19 2 N 38 N O N 20 Table 1 Inhibition of HAT by P2 proline inhibitors of the general formula H N Compound N P3 N 43 21 2 N NH 68 22 1 N N 73 23 1 N N NH2 P3 S O O N N NH O 1 Ki (nM)21 4 D-homo-Arg 13 5 D-Arg 19 6 D-Glu(OtBu) 36 7 D-Asp(OtBu) 38 8 D-Lys 40 9 D-Lys(Cbz) 53 10 D-homo-Phe 63 11 D-Val 76 12 D-Phe 98 13 D-Cha 108 14 D-Leu 120 15 D-Phe(4-Amidino) 168 16 D-Phe(4-CN) 311 17 D-Asp 1425 O 135 O N 24 1 25 1 N 149 N 158 N N 26 1 N O 183 27 1 N NH 202 28 1 N N 29 1 N N OH 210 221 4862 F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4860–4864 Table 3 Modifications in the P2 position NH H N O S O O NH2 H N P2 NH H2N Compound NH Ki (nM)21 P2 a Abu Nvab Val Ile Ala Lys(Cbz) Ser Leu Phe Asp Arg Asp(OtBu) 30 31 32 33 34 35 36 37 38 39 40 41 a a-Aminobutyric acid. b Norvaline. 14 15 22 23 30 34 34 47 57 422 457 512 Table 4 Specificity of selected compounds Compound 4 5 6 8 18 19 21 30 31 32 34 36 Ki (nM) HAT21 Thrombin28 FXa28 Plasmin28 13 19 36 40 17 38 68 14 15 22 30 34 0.39 3.5 6.7 1.0 5.2 9.8 13.5 29 68 66 74 1610 2.7 2.4 31 52 129 397 565 2.1 37 21 1.4 14.5 17.5 39 1520 63 23,130 1160 114 3030 2110 2810 590 161 expression of the proteolytic domain of TMPRSS2 in Escherichia coli and inhibition studies with these substrate analogue inhibitors will be published in due course.27 The relatively selective HAT inhibitor 31, its close analogue 30 and the most potent compound from the piperazine series (18) were selected to inhibit influenza virus propagation in cell culture. For this assay MDCK cells were used which express HAT under doxycycline-induced transcriptional activation.8 These MDCK cells were infected with various human influenza strains and incubated with the respective inhibitors for 24 h to allow multiple cycles of viral replication. Subsequently infected cells and comet-like spread of infection were immunostained using antibodies against the viral nucleoprotein (NP) as described previously.20 As expected, multicycle replication of viruses is observed in doxycycline-treated cells in the absence of inhibitors, whereas no virus spread is visible in cells lacking doxycycline-induced expression of HAT (Fig. 1). At a concentration of 1 lM (not shown) selected inhibitors and compound 5, which serves as control,8 show only a negligible effect against all three tested virus strains, whereas at 10 lM all compounds, especially inhibitors 18 and 30, strongly suppress multicycle virus replication. The synthesis of the inhibitors was performed according to previously described methods12,13,26 and is exemplarily described only for the most potent analogue 4 and its precursors 8 and 9 and for inhibitor 18 (Schemes 1 and 2). Briefly, benzylsulfonylchloride (42) was introduced to H-D-Lys(Cbz)-OH followed by coupling of H-Pro4-amidinobenzylamide  2HCl29 (44) to give inhibitor 9. Cleavage of the Cbz-group provided inhibitor 8, which was converted into the D-homo-Arg analogue 4 by reaction with 1H-pyrazole-1carboxamidine.30 Inhibitor 18 was synthesized according to Scheme 2 starting with inhibitor 6. Cleavage of the tert-butyl group provided compound 45, which was used for PyBOP mediated coupling of 1-(2pyrimidyl)-piperazine to give analogue 18. In summary, the replacement of glycine by proline in the initial inhibitor 1 improved the Ki-value by a factor of 20, but leads to poor selectivity. In fact, thrombin and factor Xa are even more inhibited than HAT by various compounds. Interestingly, the incorporation of P2 norvaline provided an inhibitor (31) with similar potency against HAT and improved selectivity against the other tested three trypsin-like serine proteases. Furthermore, this Figure 1. Inhibition of influenza virus propagation by inhibitor treatment in MDCK–HAT cells. Cells were infected with different influenza A viruses and incubated with or without doxycycline (Dox) in the presence of 10 lM of inhibitors 5, 31, 30 and 18 at 37 °C. After 24 h the cells were immunostained with influenza virus NP-specific antibodies. Infection of MDCK–HAT cells treated or not with doxycycline in the absence of inhibitors are used as control. Immunostained cells were counted using a microscope, and the percentage of infected cells compared to control (100%) is given below each well. 4863 F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4860–4864 NH 2 O H N O H 2N OH O O S O Cl NH 2 NH NH O NH OH HN S O O HN NH N O 44 42 HN H N O O S O O a b O HN O O O 9 43 NH 2 NH 2 NH O H N O S NH O NH O S O c H N O N O NH N O d NH 2 HN NH NH 2 8 4 Scheme 1. Synthesis of inhibitors 4, 8 and 9. Reagents and conditions: (a) 2.2 equiv trimethylsilylchloride, 2.2 equiv diisopropylethylamine reflux in dry dichloromethane for 1 h followed by addition of 1.1 equiv 42 and 1.1 equiv diisopropylethylamine at 0 °C for 15 min, then room temperature for 3 h, (b) 1 equiv 44, 1.1 equiv HBTU, 1.1 equiv HOBt, 3 equiv diisopropyethylamine in DMF at room temperature, 12 h, (c) 10% Pd/C, H2 atmosphere, in 90% acetic acid, room temperature for 24 h, (d) 3 equiv 1H-pyrazole1-carboxamidine hydrochloride, 4.5 equiv diisopropylethylamine in DMF, 16 h. All final compounds were purified by reversed phase HPLC to a purity of >95% according to HPLC analysis and UV detection at 220 nm and obtained as lyophilized powders. NH 2 NH 2 NH 2 NH NH O O H N O S NH N O O a O H N S O O O NH b H N S O NH N N N O O O NH O HO O N O N N 6 45 18 Scheme 2. Reagents and conditions: (a) trifluoroacetic acid for 1 h, (b) 1.1 equiv 1-(2-pyrimidyl)-piperazine, 1 equiv PyBOP, 2 equiv diisopropylethylamine, room temperature, 6 h. norvaline inhibitor 31, its close analogue 30 and compound 18 from the piperazine series were effective in suppressing replication of H1 and H3 influenza viruses in a HAT-expressing MDCK cell model. Acknowledgment The authors would like to thank Robert Etges for correcting the English version of the manuscript. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2011.06.033. References and notes 1. Taubenberger, J. K.; Morens, D. M. Public Health Rep. 2010, 125, 16. 2. Boltz, D. A.; Aldridge, J. R.; Webster, R. G.; Govorkova, E. A. Drugs 2010, 70, 1349. 3. Gong, J.; Fang, H.; Li, M.; Liu, Y.; Yang, K.; Liu, Y.; Xu, W. Curr. Med. Chem. 2009, 16, 3716. 4. Skehel, J. J.; Wiley, D. C. Annu. Rev. Biochem. 2000, 69, 531. 5. Klenk, H. D.; Garten, W. Trends Microbiol. 1994, 2, 39. 6. Böttcher, E.; Matrosovich, T.; Beyerle, M.; Klenk, H. D.; Garten, W.; Matrosovich, M. J. Virol. 2006, 80, 9896. 7. Chaipan, C.; Kobasa, D.; Bertram, S.; Glowacka, I.; Steffen, I.; Tsegaye, T. S.; Takeda, M.; Bugge, T. H.; Kim, S.; Park, Y.; Marzi, A.; Pöhlmann, S. J. Virol. 2009, 83, 3200. 8. Böttcher-Friebertshäuser, E.; Freuer, C.; Sielaff, F.; Schmidt, S.; Eickmann, M.; Uhlendorff, J.; Steinmetzer, T.; Klenk, H. D.; Garten, W. J. Virol. 2010, 84, 5605. 9. Wysocka, M.; Spichalska, B.; Lesner, A.; Jaros, M.; Brzozowski, K.; Legowska, A.; Rolka, K. Bioorg. Med. Chem. 2010, 18, 5605. 10. Szabo, R.; Bugge, T. H. Int. J. Biochem. Cell Biol. 2008, 40, 1297. 11. Gustafsson, D.; Bylund, R.; Antonsson, T.; Nilsson, I.; Nystrom, J. E.; Eriksson, U.; Bredberg, U.; Teger-Nilsson, A. C. Nat. Rev. Drug Disc. 2004, 3, 649. 12. Schweinitz, A.; Stürzebecher, A.; Stürzebecher, U.; Schuster, O.; Stürzebecher, J.; Steinmetzer, T. Med. Chem. 2006, 2, 349. 13. Schweinitz, A.; Steinmetzer, T.; Banke, I. J.; Arlt, M. J.; Stürzebecher, A.; Schuster, O.; Geissler, A.; Giersiefen, H.; Zeslawska, E.; Jacob, U.; Kruger, A.; Stürzebecher, J. J. Biol. Chem. 2004, 279, 33613. 14. Bajusz, S.; Szell, E.; Bagdy, D.; Barabas, E.; Horvath, G.; Dioszegi, M.; Fittler, Z.; Szabo, G.; Juhasz, A.; Tomori, E.; Szilagyi, G. J. Med. Chem. 1990, 33, 1729. 4864 F. Sielaff et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4860–4864 15. Tucker, T. J.; Lumma, W. C.; Mulichak, A. M.; Chen, Z.; Naylor-Olsen, A. M.; Lewis, S. D.; Lucas, R.; Freidinger, R. M.; Kuo, L. C. J. Med. Chem. 1997, 40, 830. 16. Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. Bioinformatics 2006, 22, 195. 17. Kiefer, F.; Arnold, K.; Kunzli, M.; Bordoli, L.; Schwede, T. Nucleic Acids Res. 2009, 37, D387. 18. Peitsch, M. C. Nature Biotechnol. 1995, 13, 658. 19. Kyrieleis, O. J.; Huber, R.; Ong, E.; Oehler, R.; Hunter, M.; Madison, E. L.; Jacob, U. FEBS J. 2007, 274, 2148. 20. Böttcher, E.; Freuer, C.; Steinmetzer, T.; Klenk, H. D.; Garten, W. Vaccine 2009, 27, 6324. Compounds 1–3 are designated as SPI-1-3 in this publication. 21. The inhibition constants were determined with recombinant human airway trypsin-like protease (R&D Systems) at RT according to the method of Dixon using a Safire2 fluorescence plate reader (Tecan) (kex = 380 nm; kem = 460 nm) and D-cyclohexylalanine-Pro-Arg-AMC as the substrate in 50 mM Tris buffer (pH 9.5) containing 0.05% Brij 58 and 1 mg/mL BSA. The enzyme concentration used in the assay was 23.8 pM, and the substrate concentrations were 50, 100 and 200 lM. Results were obtained from at least two independent experiments. 22. The synthesis for inhibitors 5 and 13 was previously described at Sisay et al J. Med. Chem. 2010, 53, 5523. 23. Bajusz, S.; Barabas, E.; Tolnay, P.; Szell, E.; Bagdy, D. Int. J. Pept. Protein Res. 1978, 12, 217. 24. Gustafsson, D.; Antonsson, T.; Bylund, R.; Eriksson, U.; Gyzander, E.; Nilsson, I.; Elg, M.; Mattsson, C.; Deinum, J.; Pehrsson, S.; Karlsson, O.; Nilsson, A.; Sörensen, H. Thromb. Haemost. 1998, 79, 110. 25. Hellstern, P.; Stürzebecher, U.; Wuchold, B.; Haubelt, H.; Seyfert, U. T.; Bauer, M.; Vogt, A.; Stürzebecher, J. J. Thromb. Haemost. 2007, 5, 2119. 26. Sisay, M. T.; Steinmetzer, T.; Stirnberg, M.; Maurer, E.; Hammami, M.; Bajorath, J.; Gütschow, M. J. Med. Chem. 2010, 53, 5523. 27. The catalytic domain of TMPRSS2 was expressed in E. coli according to methods described previously for matriptase (Steinmetzer et al., J. Med. Chem. 2006, 49, 4116). The inhibition constants were determined with the recombinant expressed catalytic domain at RT according to the method of Dixon using a Safire2 flourescence plate reader (Tecan) (kEx = 380 nm, kEm = 460 nm) and Dcyclohexylalanine-Pro-Arg-AMC as the substrate in 50 mM Tris-buffer (pH 8.0, 154 mM NaCl). For Ki-value determinations three different substrate concentrations were used (50, 100, and 200 lM). Results were obtained from at least two independent experiments. 28. Stürzebecher, J.; Prasa, D.; Hauptmann, J.; Vieweg, H.; Wikström, P. J. Med. Chem. 1997, 40, 3091. 29. Steinmetzer, T.; Nowak, G. Patent No. WO 02/059065, 2002. 30. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. J. Org. Chem. 1992, 57, 2497.