Novel RU486 (mifepristone) analogues with increased activity against Venezuelan Equine Encephalitis Virus but reduced progesterone receptor antagonistic activity

There are currently no therapeutics to treat infection with the alphavirus Venezuelan equine encephalitis virus (VEEV), which causes flu-like symptoms leading to neurological symptoms in up to 14% of cases. Large outbreaks of VEEV can result in 10,000 s of human cases and mass equine death. We previously showed that mifepristone (RU486) has anti-VEEV activity (EC50 = 20 μM) and only limited cytotoxicity (CC50 > 100 μM), but a limitation in its use is its abortifacient activity resulting from its ability to antagonize the progesterone receptor (PR). Here we generate a suite of new mifepristone analogues with enhanced antiviral properties, succeeding in achieving >11-fold improvement in anti-VEEV activity with no detectable increase in toxicity. Importantly, we were able to derive a lead compound with an EC50 of 7.2 µM and no detectable PR antagonism activity. Finally, based on our SAR analysis we propose avenues for the further development of these analogues as safe and effective anti-VEEV agents.

[1]  R. Rico-Hesse,et al.  Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[2]  G. Teutsch,et al.  11β-substituted steroids, an original pathway to antihormones , 1988 .

[3]  D. Philibert,et al.  New analogues of mifepristone with more dissociated antiprogesterone activities. , 1989, Journal of steroid biochemistry.

[4]  D. Weiner,et al.  Phase I/II Trial of the Anti-HIV Activity of Mifepristone in HIV-Infected Subjects ACTG 5200 , 2010, Journal of acquired immune deficiency syndromes.

[5]  D. Harrich,et al.  Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus , 2012, The Biochemical journal.

[6]  M. Fornerod,et al.  Venezuelan Equine Encephalitis Virus Capsid Protein Forms a Tetrameric Complex with CRM1 and Importin α/β That Obstructs Nuclear Pore Complex Function , 2010, Journal of Virology.

[7]  Li Hai,et al.  A new and efficient method for the synthesis of Ulipristal acetate , 2014, Steroids.

[8]  A. Leusen,et al.  Chemistry of sulfonylmethyl isocyanides. 13. A general one-step synthesis of nitriles from ketones using tosylmethyl isocyanide. Introduction of a one-carbon unit , 1977 .

[9]  Regio and stereospecific synthesis of 11β substituted 19 norsteroids , 1979 .

[10]  R. Rico-Hesse,et al.  Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America , 1996, The Lancet.

[11]  S. Paessler,et al.  Rapid, non-invasive imaging of alphaviral brain infection: reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals. , 2011, Vaccine.

[12]  S. Fix,et al.  Synthesis and antihormonal properties of novel 11β-benzoxazole-substituted steroids. , 2012, Bioorganic & medicinal chemistry letters.

[13]  R. Barrera,et al.  Venezuelan equine encephalitis. , 2003, Annual review of entomology.

[14]  W. Siegert,et al.  Guideline to reference gene selection for quantitative real-time PCR. , 2004, Biochemical and biophysical research communications.

[15]  G. Allan,et al.  Synthesis and identification of novel 11beta-aryl-4',5'-dihydrospiro[estra-4,9-diene-17beta,4'-oxazole] analogs with dissociated antiprogesterone activities. , 2007, Bioorganic & medicinal chemistry letters.

[16]  K. Kehn-Hall,et al.  Nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce Venezuelan Equine Encephalitis Virus replication. , 2013, Antiviral research.

[17]  D. Martin,et al.  Venezuelan equine encephalitis. , 1974, Advances in veterinary science and comparative medicine.

[18]  Jillian T Henderson,et al.  Safety of mifepristone abortions in clinical use. , 2005, Contraception.

[19]  P. Saunders,et al.  Selective progesterone receptor modulators (SPRMs): progesterone receptor action, mode of action on the endometrium and treatment options in gynecological therapies , 2016, Expert Opinion on Therapeutic Targets.

[20]  P. N. Rao,et al.  A practical large-scale synthesis of 17α-acetoxy-11β-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione (CDB-2914) , 2000, Steroids.

[21]  Bernard Perbal,et al.  Ten years later… , 2009, Journal of Cell Communication and Signaling.

[22]  Gert Vriend,et al.  YASARA View—molecular graphics for all devices—from smartphones to workstations , 2014, Bioinform..

[23]  K. Kehn-Hall,et al.  Novel inhibitors targeting Venezuelan equine encephalitis virus capsid protein identified using In Silico Structure-Based-Drug-Design , 2017, Scientific Reports.

[24]  E. Frolova,et al.  Venezuelan Equine Encephalitis Virus Variants Lacking Transcription Inhibitory Functions Demonstrate Highly Attenuated Phenotype , 2014, Journal of Virology.

[25]  Bharathi,et al.  Aryltitanium species through the reaction of N,N-dialkylarylamines with TiCl(4): oxidative coupling, N-dealkylation, and reaction with electrophiles , 2000, The Journal of organic chemistry.

[26]  R. Garfield,et al.  Synthesis and biological evaluation of partially fluorinated antiprogestins and mesoprogestins , 2013, Steroids.

[27]  Colin W Pouton,et al.  Mechanism of Microtubule-facilitated “Fast Track” Nuclear Import* , 2011, The Journal of Biological Chemistry.

[28]  K. Koehler,et al.  Liver-selective glucocorticoid antagonists: a novel treatment for type 2 diabetes. , 2004, Journal of medicinal chemistry.

[29]  K. Kehn-Hall,et al.  Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells , 2016, PLoS neglected tropical diseases.

[30]  O. Radostits.  Advances in Veterinary Science and Comparative Medicine , 1983 .

[31]  K. Kehn-Hall,et al.  Identification of novel antivirals inhibiting recognition of Venezuelan equine encephalitis virus capsid protein by the Importin &agr;/&bgr;1 heterodimer through high‐throughput screening , 2018, Antiviral research.

[32]  J. Shine,et al.  Progestin Inhibition of Progesterone Receptor Gene Expression in Human these steroids is determined in part by the cellular Breast Cancer Cells , 1989 .

[33]  D. Jans,et al.  An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import , 2011, Journal of biomolecular screening.

[34]  J. Fagart,et al.  Molecular determinants of the recognition of ulipristal acetate by oxo-steroid receptors , 2014, The Journal of Steroid Biochemistry and Molecular Biology.

[35]  Susan A Charman,et al.  Methods to assess drug permeability across the blood‐brain barrier , 2006, The Journal of pharmacy and pharmacology.

[36]  N. Chabbert-Buffet,et al.  Selective progesterone receptor modulators: current applications and perspectives , 2018 .

[37]  U. Klar,et al.  Beneficial effects of fluorine in the anti-progestin ZK 230211 , 2005 .

[38]  C. Libert,et al.  Selective glucocorticoid receptor modulation: New directions with non-steroidal scaffolds. , 2015, Pharmacology & therapeutics.

[39]  A. Lapi,et al.  N-demethylation of N,N-dimethylanilines by the benzotriazole N-oxyl radical: evidence for a two-step electron transfer-proton transfer mechanism. , 2010, The Journal of organic chemistry.

[40]  S. Sampathi,et al.  In Silico, In Vitro and In Vivo Anti-inflammatory and Analgesic Activity of Usnic Acid , 2019 .

[41]  R. C. Macridis.  A review , 1963 .

[42]  S. Weaver,et al.  Analysis of Venezuelan Equine Encephalitis Virus Capsid Protein Function in the Inhibition of Cellular Transcription , 2007, Journal of Virology.

[43]  M. Laufgraben,et al.  Mifepristone for Management of Cushing's Syndrome , 2013, Pharmacotherapy.

[44]  R. Rico-Hesse,et al.  Association of Venezuelan equine encephalitis virus subtype IE with two equine epizootics in Mexico. , 1998, The American journal of tropical medicine and hygiene.

[45]  S. Scholz,et al.  Synthese von 14,17‐überbrückten 11β‐Arylsteroiden , 1989 .

[46]  M. Bogoyevitch,et al.  Cytokine‐Induced Slowing of STAT3 Nuclear Import; Faster Basal Trafficking of the STAT3β Isoform , 2014, Traffic.

[47]  R. Steiner,et al.  A novel, high-affinity, fluorescent progesterone receptor antagonist. Synthesis and in vitro studies. , 2004, Bioconjugate chemistry.

[48]  D. Jans,et al.  Molecular dissection of an inhibitor targeting the HIV integrase dependent preintegration complex nuclear import , 2018, Cellular microbiology.

[49]  J. Graham,et al.  Progesterone stimulates progenitor cells in normal human breast and breast cancer cells , 2014, Breast Cancer Research and Treatment.

[50]  Meihua Rose Feng,et al.  Assessment of blood-brain barrier penetration: in silico, in vitro and in vivo. , 2002, Current drug metabolism.

[51]  Narasimhan J. Venkatachari,et al.  Antiviral effects of mifepristone on human immunodeficiency virus type-1 (HIV-1): targeting Vpr and its cellular partner, the glucocorticoid receptor (GR). , 2006, Antiviral research.

[52]  D. Edwards,et al.  Progesterone receptor transcription and non-transcription signaling mechanisms , 2003, Steroids.

[53]  E. Baulieu,et al.  RU486 (mifepristone): mechanisms of action and clinical uses. , 1997, Annual review of medicine.

[54]  D. Edwards,et al.  16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[55]  C. Clarke,et al.  Effect of overexpression of progesterone receptor A on endogenous progestin-sensitive endpoints in breast cancer cells. , 1999, Molecular endocrinology.

[56]  A. Al-Hendy,et al.  Selective progesterone receptor modulators for fertility preservation in women with symptomatic uterine fibroids† , 2017, Biology of Reproduction.

[57]  Peter Ertl,et al.  Bioisosteric Replacement and Scaffold Hopping in Lead Generation and Optimization , 2010, Molecular informatics.

[58]  S. London,et al.  Mifepristone (RU486): a review. , 1997, Fertility and sterility.

[59]  O. Heikinheimo,et al.  The pharmacokinetics of mifepristone in humans reveal insights into differential mechanisms of antiprogestin action. , 2003, Contraception.

[60]  A. D. Cross,et al.  Spectra and Stereochemistry. VII.1,2 Long-Range Shielding by Nitrile Groups , 1963 .

[61]  E. Frolova,et al.  Interplay of Acute and Persistent Infections Caused by Venezuelan Equine Encephalitis Virus Encoding Mutated Capsid Protein , 2010, Journal of Virology.

[62]  G. Alvisi,et al.  Global enhancement of nuclear localization‐dependent nuclear transport in transformed cells , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[63]  Robert P. Sheridan,et al.  The Most Common Chemical Replacements in Drug-Like Compounds , 2002, J. Chem. Inf. Comput. Sci..

[64]  E. Frolova,et al.  Venezuelan Equine Encephalitis Virus Capsid Protein Inhibits Nuclear Import in Mammalian but Not in Mosquito Cells , 2008, Journal of Virology.

[65]  Li Hai,et al.  A simple and convenient synthetic route to Ulipristal acetate , 2013, Steroids.

[66]  K. Horwitz,et al.  Differential Gene Regulation by the Two Progesterone Receptor Isoforms in Human Breast Cancer Cells* , 2002, The Journal of Biological Chemistry.

[67]  E. Schaff.  Mifepristone: ten years later. , 2010, Contraception.

[68]  Jonathan Greer,et al.  The Three-dimensional Structures of Antagonistic and Agonistic Forms of the Glucocorticoid Receptor Ligand-binding Domain , 2003, Journal of Biological Chemistry.