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EP3876927A1 - Strigolactones destinées à être utilisées dans la prévention et/ou le traitement d'infections causées par des virus de la famille des herpesviridae - Google Patents

Strigolactones destinées à être utilisées dans la prévention et/ou le traitement d'infections causées par des virus de la famille des herpesviridae

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Publication number
EP3876927A1
EP3876927A1 EP19831876.8A EP19831876A EP3876927A1 EP 3876927 A1 EP3876927 A1 EP 3876927A1 EP 19831876 A EP19831876 A EP 19831876A EP 3876927 A1 EP3876927 A1 EP 3876927A1
Authority
EP
European Patent Office
Prior art keywords
viruses
cells
use according
compound
herpesviridae family
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19831876.8A
Other languages
German (de)
English (en)
Inventor
Santo Landolfo
Marco DE ANDREA
Valentina DELL'OSTE
Matteo BIOLATTI
Cristina PRANDI
Marco BLANGETTI
Emma ARTUSO
Chiara LOMBARDI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universita degli Studi di Torino
Original Assignee
Universita degli Studi di Torino
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Publication date
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Publication of EP3876927A1 publication Critical patent/EP3876927A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

  • the present invention concerns strigolactones for use in preventing and/or treating infections caused by viruses of the Herpesviridae family.
  • Strigolactones are a class of signalling molecules produced by the roots of the majority of higher plants. They play a dual role, endogenous and exogenous. At endogenous level they represent a new class of vegetable hormones which intervene in regulating the development of the root system and foliage and in general of the plant in relation to the nutritional conditions (deficiency of nutrients, in particular phosphates) . At exogenous level they represent chemical signals that are released in the rhizosphere and are perceived by microorganisms that live in association with the plant, with beneficial effects on the establishment of symbiosis.
  • Herpesviruses are viruses with double-strand DNA responsible for infections, including serious infections in humans.
  • Herpes Simplex virus type 1 HSV-1
  • Herpes Simplex virus type 2 HSV-2
  • Varicella-Zoster virus VZV
  • Cytomegalovirus CMV
  • human herpesvirus 6 human herpesvirus 7
  • Epstein-Barr Virus EBV
  • human herpesvirus 8 or Kaposi's sarcoma-associated herpesvirus, KSHV
  • the antiviral drugs of choice for herpes infections entail the use of nucleoside analogues (for example, ganciclovir) .
  • nucleoside analogues can have significant toxicity for the host.
  • the use of ganciclovir or similar nucleoside derivatives in children (congenital infections or bone marrow transplant) due to its high myelotoxicity, can have serious side effects which significantly limit its use.
  • the nucleoside analogues frequently induce the appearance of resistance mutations at gene level which encode for the viral enzymes assigned to synthesis of the DNA.
  • the object of the present invention is therefore to provide new antiviral compounds for use in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family, in particular HCMV, which overcome the above- mentioned problems.
  • said object is achieved by use of the compounds as defined in claim 1 in preventing and/or treating infections caused by viruses of the Herpesviridae family.
  • FIG. 1A shows the results of an MTT colorimetric assay (3- (4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyltetrazolium bromide; Sigma Aldrich) on human primary fibroblasts isolated from foreskin (HFF) (left-hand panel) and on VERO (right-hand panel) to evaluate the cytotoxicity of the molecules TH-EGO, EDOT, EGO-10, GR24 ;
  • MTT colorimetric assay 3- (4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyltetrazolium bromide; Sigma Aldrich
  • Figure IB shows graphs of the viral titration of the supernatants obtained from cells treated with the different strigolactones or equal volume of solvent (DMSO) at the concentration of 12.5 mM and infected with HSV-1/2 (MOI 0.1) (central and right-hand panel respectively) ;
  • FIG. 2A shows the results of an MTT colorimetric assay on HFF cells (left-hand panel) and on VERO (right-hand panel) to evaluate the cytotoxicity of the molecules TH-ABC, EDOT-ABC;
  • FIG. 2B shows graphs of the dose-response titration of the viral supernatants obtained from cells treated with the different strigolactones or equal volume of solvent (DMSO) and infected with HCMV (MOI 0.1) (upper panel) and the dose- response titration of the viral supernatants obtained from cells treated with the different strigolactones or equal volume of solvent (DMSO) and infected with HSV-1 and HSV-2 (MOI 0.1) (lower panels);
  • FIG. 3A shows the results of attachment assays (left-hand panel) and entry assays (right-hand panel) to evaluate the inhibitory activity of the molecules during the phases of attachment or penetration of the virus (HCMV) vis-a-vis the host cell;
  • FIG. 3B illustrates a Western blot of infection kinetics (MOI 0.1) 24, 48 and 72 hpi on HFF cells treated with TH-EGO or EDOT at the dose of 12.5 mM;
  • Figure 4A illustrates the evaluation of the necrosis and cell apoptosis by labelling with propidium iodide (PI) and with annexin V in cells (HFF) treated for 24 and 48 hours with TH-EGO, EDOT, DMSO at the concentration of 12.5 mM in the absence of HCMV (left-hand upper and lower panel) or presence of HCMV (right-hand upper and lower panel);
  • PI propidium iodide
  • HFF annexin V in cells
  • FIG. 4B illustrates the results of an enzymatic assay to evaluate the activity of the caspase-3 in cells (HFF) treated for 24 hours with TH-EGO, EDOT, DMSO at the concentration of 12.5 mM in the absence or presence of HCMV.
  • Ri is a substituted or unsubstituted linear or branched C1-C4 alkyl
  • R 2 is selected from the group consisting of H, substituted or unsubstituted phenyl, bromine, heteroaryl, thiophene and functionalised thiophene, are used in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family.
  • Ri is preferably CH3.
  • R2 is preferably in position 7.
  • R2 is preferably thiophene or ethylenedioxythiophene .
  • the viruses of the Herpesviridae family are Cytomegalovirus, Herpes Simplex virus 1 or Herpes Simplex virus 2, even more preferably Cytomegalovirus.
  • compositions comprising the above-mentioned compounds are furthermore used in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family.
  • the above-mentioned compositions comprise an isomer of the compounds, a salt or a pharmaceutically permitted mixture, additives and/or excipients.
  • the above- mentioned compositions comprise at least a cyclodextrin derivative, for example nanosponges or reticulated cyclodextrins .
  • the above-mentioned compounds or compositions are used preferably in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family in paediatric patients with congenital infections or who have undergone a bone marrow transplant, or in adult patients who have undergone a transplant.
  • strigolactones do not influence the initial phases of interaction of the virus in the target cells, but act in the later phases of viral replication;
  • Example 1 synthesis and characterization of strigolactone analogues .
  • the structural analogues of the strigolactones subject of this study are shown below in Table 1.
  • the Table shows: the structure of the strigolactones (SL) (first left-hand column), the abbreviation (central column) and the IUPAC name (left-hand column) .
  • the molecules 1-4 were used as racemic mixtures.
  • the molecules indicated as TH-EGO, EDOT, EGO-10 were used as racemic mixtures and were synthesized according to the procedure reported in Prandi et al . (2011), European Journal of Organic Chemistry, 3781-3793.
  • GR24 also used as a racemic mixture, was purchased from Strigolab srl.
  • TH-ABC and EDOT-ABC were synthesized according to the procedure reported in Prandi et al . (2011), with the exception of the last synthetic passage.
  • Example 2 the strigolactones inhibit the replication of the human Cytomegalovirus and Herpes Simplex virus type 1 and 2.
  • HCMV human Cytomegalovirus
  • HFF human foreskin fibroblasts
  • VEO African Green Monkey cells
  • HFF and VERO were grown in DMEM (Dulbecco's Modified Eagle Medium, SIGMA-ALDRICH) , in the presence of 10% fetal bovine serum (FBS), glutamine 2 mM, sodium pyruvate 1 mM, penicillin 100 U/mL and streptomycin sulphate 100 pg/rnL (SIGMA-ALDRICH) .
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • glutamine 2 mM glutamine 2 mM
  • sodium pyruvate 1 mM penicillin 100 U/mL
  • streptomycin sulphate 100 pg/rnL SIGMA-ALDRICH
  • the toxicity of the SL vis-a-vis the HFF and VERO was determined, using the MTT colorimetric test ( 3— ( 4 , 5— dimethylthiazol-2-yl ) -2 , 5-diphenyltetrazolium bromide; SIGMA- ALDRICH) .
  • MTT colorimetric test 3— ( 4 , 5— dimethylthiazol-2-yl ) -2 , 5-diphenyltetrazolium bromide; SIGMA- ALDRICH
  • the cells were exposed to increasing concentrations of molecule or dimethyl sulfoxide (DMSO) carrier for an incubation time of 144 hours (h) .
  • DMSO dimethyl sulfoxide
  • the HFF cells were infected with HCMV, at a multiplicity of infection (MOI) of 0.1, and the VERO cells with HSV-1 or HSV-2 at MOI of 0.1.
  • the cells were treated one hour prior to the infection and for the entire duration of the infection with the SL (12.5 mM, a non-toxic dose for the cells as can be seen from the cell cytotoxicity test) or with equal volume of DMSO solvent .
  • Titration of the virus present in the supernatants of the samples treated as described previously was carried out by means of plaque assay. More specifically, the supernatant of the samples under examination and the cells were collected and lysed via three nitrogen/37 °C cycles and subsequently centrifuged at 1500 rpm for 10 min to eliminate the cell debris. The supernatant thus obtained, containing the viral particles, was analysed via plaque assay for determination of the viral titre.
  • HFF or VERO cells grown in plates with 96 wells in 100 m ⁇ of DMEM at 10% of FBS, were infected with serial dilutions of the virus and centrifuged at 2000 rpm for 30 minutes (min), to promote adsorption.
  • the viral inoculum was removed and replaced with 100 m ⁇ of methylcellulose 0.8% ( SIGMA-ALDRICH) , 1% FBS.
  • SIGMA-ALDRICH methylcellulose 0.8%
  • FBS methylcellulose 0.8%
  • the methylcellulose was removed and substituted with a solution of crystal violet 0.1% diluted in ethanol 10% (SIGMA- ALDRICH) for 30 min in the dark.
  • the plate was then decolorized under running water and the infection plaques counted under the inverted optical microscope (LEIKA DMIL) .
  • TH-EGO and EDOT were the most promising molecules, as they were characterised by an inhibitory activity against HCMV, HSV-1 and HSV-2 greater than EGO-10 and GR24; they were therefore used for the subsequent experiments .
  • Example 3 the derivatives of TH-EGO and EDOT effectively inhibit the replication of HCMV and HSV-1 and HSV-2.
  • TH-EGO and EDOT proved to be the compounds that most effectively inhibit the replication of HCMV, HSV-1 and HSV-2.
  • the derivatives TH-ABC and EDOT-ABC were synthesized which, compared to TH-EGO and EDOT, respectively, do not have the enol ether bridge bound to the butenolide ring .
  • the HFF cells were infected with HCMV at MOI of 0.1, and the VERO cells with HSV-1 or HSV-2 at MOI of 0.1.
  • the cells were treated with scalar concentrations of molecule, or with equal volume of DMSO solvent, one hour prior to the infection and for the entire duration of the infection.
  • the virus was collected and titred from the samples treated 144 h after infection with HCMV and 48 hours after infection with HSV-1 and HSV-2 as described in the previous paragraph (Fig. 2B) .
  • the different SL analysed highlighted a dose-dependent inhibitory activity against HCMV, HSV-1 and HSV-2.
  • the IC50 values (inhibiting concentration necessary to inhibit 50% of the viral replication) of TH-EGO and EDOT are given below .
  • Example 4 determination of the antiviral activity of the SL on HCMV.
  • HFF target cell
  • the cells were fixed, coloured with a solution of crystal violet and the viral plaques were counted.
  • the number of plaques obtained in the sample treated with the carrier at maximum dose was set to 100%. From the data obtained, it can be seen that the SL do not significantly inhibit the process of attachment of HCMV to the target cell (Fig. 3A) .
  • the HFF cells were cooled beforehand to 4°C, infected with HCMV (MOI 0.1), kept at 4°C for 3 h and lastly treated with scalar concentrations of SL or DMSO and incubated for 3 h at 37 °C to allow entry of the virus.
  • the cells were treated with acid glycine (100 mM glycine, 150 mM NaCl pH 3) to remove the virus that had not penetrated into the cells.
  • acid glycine 100 mM glycine, 150 mM NaCl pH 3
  • the cells were fixed, coloured with a solution of crystal violet and the viral plaques were counted.
  • the number of plaques obtained in the sample treated with the carrier at maximum dose was set to 100%. From the data obtained it can be seen that the SL analysed do not significantly alter the process of entry of HCMV into the target cell (Fig. 3A) .
  • the cell protein a-Tubulin was included as an internal loading control.
  • the HFF cells were infected with HCMV at MOI 0.3. These cultures were treated one hour prior to infection and for the entire duration of the infection with 12.5 mM of TH-EGO or EDOT, and the control cultures with the same volume of DMSO solvent. The cells were then collected at 24, 48 and 72 h from the infection (hpi, "hours post infection") .
  • the cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Nonidet P-40; 0.1% SDS; 0.5% deoxycholate ; protease inhibitors) and then quantified with the Bradford method (BIO-RAD) .
  • RIPA buffer 50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Nonidet P-40; 0.1% SDS; 0.5% deoxycholate ; protease inhibitors
  • 30 pg of protein extract were collected, to which an appropriate quantity of Laemmli sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 2% b-mercaptoethanol ) was added.
  • the solution thus obtained was heated to 95°C for 5 minutes and separated by means of electrophoresis on polyacrylamide gel (8%) -SDS page.
  • the gel was analysed using the ChemiDoc (BIORAD) instrument. From the results obtained, it can be seen that the inhibition of the viral replication occurs during the late stage of the infection, since a reduction is found in the expression levels of the proteins UL44 and pp28 (Fig. 3B) .
  • Example 5 virolysis as an antiviral activity of the SL.
  • HFF cells were treated with SL (12.5 mM) , one hour prior to infection and for the entire duration of the infection, or with equal volume of DMSO solvent and infected with HCMV (MOI 1) . After 24 and 48 hours the cells were processed by means of Annexin V-FITC Apoptosis Detection Kit (Calbiochem) , a technique which highlights the presence of apoptotic and necrotic cells through cytofluorimetric analysis .
  • PS phosphatidylserine
  • FITC conjugated fluorophore
  • the fluorophore propidium iodide is added, a DNA intercalating agent, which intercalates stoichiometrically and emits in the red spectral region if the cell membrane is not intact, thus discriminating between live cells (negative annexin and propidiumn iodide) , apoptotic cells (positive annexin, negative propidium) and necrotic cells (negative annexin, positive propidium) .
  • the cytofluorimetric analysis was performed with the FACSCalibur flow cytometer (BD Biosciences) (excitation at 488 nm, emission 518 nm and 400 nm for FITC and propidium respectively) and the results were analysed with the program ModFit LT software (BD Biosciences) .
  • HFF cells were treated for 24 h with the SL TH-EGO, EDOT, or with the solvent DMSO at a concentration of 12.5 mM, in the absence or presence of HCMV (MOI 1) and subsequently analysed by means of the fluorimetric assay SensoLyte AFC Caspase Sampler Kit Fluorimetric (Anaspec, CA, USA) , which measures the cleavage of a fluorescent substrate of the enzyme caspase-3.
  • the fluorescence intensity was measured with the Victor3 multi-plate reader spectrophotometer (Perkin Elmer) (excitation 405 nm, emission 500 nm) .
  • the RFU values relative units of fluorescence obtained from the treated cells were expressed as fold induction with respect to the non-infected sample treated only with DMSO solvent, the value of which was set equal to 1.
  • the results obtained indicate that the exposure of the HFF cells to the SL and to the infection caused by HCMV significantly induces the activity of the caspase-3 enzyme with respect to the controls (Fig. 4B) .

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
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  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
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Abstract

La présente invention concerne des composés de formule (I) dans laquelle R1 représente un alkyle en C1-C4 linéaire ou ramifié substitué ou non substitué, et R2 est choisi dans le groupe constitué par H, un phényle substitué ou non substitué, un brome, un hétéroaryle, un thiophène et un thiophène fonctionnalisé, à utiliser dans la prévention et/ou le traitement d'infections provoquées par des virus de la famille des Herpesviridae.
EP19831876.8A 2018-11-08 2019-11-08 Strigolactones destinées à être utilisées dans la prévention et/ou le traitement d'infections causées par des virus de la famille des herpesviridae Withdrawn EP3876927A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT201800010142 2018-11-08
PCT/IB2019/059611 WO2020095260A1 (fr) 2018-11-08 2019-11-08 Strigolactones destinées à être utilisées dans la prévention et/ou le traitement d'infections causées par des virus de la famille des herpesviridae

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EP3876927A1 true EP3876927A1 (fr) 2021-09-15

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EP19831876.8A Withdrawn EP3876927A1 (fr) 2018-11-08 2019-11-08 Strigolactones destinées à être utilisées dans la prévention et/ou le traitement d'infections causées par des virus de la famille des herpesviridae

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EP (1) EP3876927A1 (fr)
WO (1) WO2020095260A1 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2581081A3 (fr) * 2007-06-01 2013-07-31 The Trustees Of Princeton University Traitement d'infections virales par modulation de voies métaboliques de cellules hôtes
WO2010125065A2 (fr) * 2009-04-28 2010-11-04 Bayer Cropscience Ag Compositions comprenant un composé strigolactone et un composé chito-oligosaccharide pour une croissance végétale et un rendement végétal accrus
CA2849343C (fr) * 2011-09-21 2020-12-15 The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) Utilisation de strigolactones et d'analogues de strigolactone pour traiter des affections proliferatives

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