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Id Subject Object Predicate Lexical cue
T1 0-97 Sentence denotes Discovery of a Bradykinin B2 Partial Agonist Profile of Raloxifene in a Drug Repurposing Campaign
T2 99-107 Sentence denotes Abstract
T3 108-328 Sentence denotes Covid-19 urges a deeper understanding of the underlying molecular mechanisms involved in illness progression to provide a prompt therapeutical response with an adequate use of available drugs, including drug repurposing.
T4 329-470 Sentence denotes Recently, it was suggested that a dysregulated bradykinin signaling can trigger the cytokine storm observed in patients with severe Covid-19.
T5 471-643 Sentence denotes In the scope of a drug repurposing campaign undertaken to identify bradykinin antagonists, raloxifene was identified as prospective compound in a virtual screening process.
T6 644-817 Sentence denotes The pharmacodynamics profile of raloxifene towards bradykinin receptors is reported in the present work, showing a weak selective partial agonist profile at the B2 receptor.
T7 818-939 Sentence denotes In view of this new profile, its possible use as a therapeutical agent for the treatment of severe Covid-19 is discussed.
T8 941-943 Sentence denotes 1.
T9 944-956 Sentence denotes Introduction
T10 957-1253 Sentence denotes About 20% of patients infected with SARS-Cov-2 develop respiratory complications, including pneumonia, that may evolve to severe hypoxemia, acute respiratory distress syndrome (ARDS), pulmonary edema, shock, metabolic acidosis, coagulation dysfunction and multiple organ functional failure [1,2].
T11 1254-1438 Sentence denotes Covid-19 patients with severe symptoms exhibit an immune response characterized by elevated serum levels of diverse proinflammatory molecules, including interleukin-6 (IL-6) [3,4,5,6].
T12 1439-1773 Sentence denotes Presently, these patients are treated with anti-inflammatory drugs like dexamethasone [7] or cytokine inhibitors, combined with antibiotics to treat secondary infections, sometimes combined with remdesivir, an antiviral originally developed to treat hepatitis C and tested against the Ebola virus disease to reduce the viral load [8].
T13 1774-2007 Sentence denotes Treatment of Covid-19 urges a deeper understanding of the underlying molecular mechanisms involved in disease progression to provide a prompt therapeutical response with an adequate use of available drugs, including drug repurposing.
T14 2008-2129 Sentence denotes Indeed, there is a number of ongoing clinical trials to test the efficacy of diverse FDA-approved drugs against Covid-19.
T15 2130-2276 Sentence denotes Among them, a few involve assessment of the therapeutic efficacy of IL-6 inhibition, unfortunately without reporting conclusive results [9,10,11].
T16 2277-2446 Sentence denotes Recently, it was proposed that dysregulated bradykinin (BK) signaling could be the trigger of the cytokine storm observed in patients with severe Covid-19 [12,13,14,15].
T17 2447-2682 Sentence denotes The rational for this hypothesis relies on the increased availability of BK and its metabolite des-Arg9-BK in Covid-19 patients, due to the downregulation of angiotensin converting enzyme 2 (ACE2), the entry protein of SARS-Cov-2 [16].
T18 2683-2864 Sentence denotes Indeed, ACE2 downregulation increases the amount of angiotensin I processed by the angiotensin-converting enzyme (ACE) with a concomitant decrease of its degradation activity on BK.
T19 2865-3001 Sentence denotes Moreover, levels of des-Arg9-BK are also increased due to the diminished degradative activity of ACE2 caused by its downregulation [12].
T20 3002-3102 Sentence denotes The BK hypothesis is consistent with the observed elevated levels of IL-6 in Covid-19 patients [17].
T21 3103-3240 Sentence denotes This new perspective suggests that inhibition of BK signaling may be a suitable therapy to avoid the cytokine storm and its consequences.
T22 3241-3453 Sentence denotes Indeed, as a proof of principle, preliminary results from a limited study suggests that inhibition of the BK signaling is associated with a decrease of the oxygen supplementation needed by Covid-19 patients [18].
T23 3454-3659 Sentence denotes Nowadays, icatibant is the only BK antagonist approved as therapeutic agent for the treatment of hereditary angioedema [19], in spite of the number of selective BK ligands described in the literature [20].
T24 3660-3832 Sentence denotes The discovery of the bradykinin hypothesis prompted the launch of an ongoing clinical trial to assess the benefits of icatibant for the treatment of covid-19 patients [21].
T25 3833-4023 Sentence denotes In other to swiftly test the efficacy of alternative BK antagonists with diverse pharmacological profiles for the treatment of Covid-19, drug repurposing represents a valuable strategy [22].
T26 4024-4194 Sentence denotes To this aim, we recently carried out a virtual screening study on the DrugBank database [23] aimed at identifying BK ligands among drugs already approved by the FDA [24].
T27 4195-4385 Sentence denotes The study was carried out using four points common to the pharmacophores derived for the B1 and B2 bradykinin receptors recently reported that were used as queries for the search [25,26,27].
T28 4386-4510 Sentence denotes Hits identified were subsequently subjected to a docking study using the Molecular Operating Environment (MOE) program [28].
T29 4511-4662 Sentence denotes The virtual screening process permitted the identification of a small set of drugs as prospective nonselective BK ligands, as described elsewhere [24].
T30 4663-4831 Sentence denotes Specifically, the DrugBank database, containing a total of 1703 molecules, was screened for compounds that fulfilled a common pharmacophore for the B1 and B2 receptors.
T31 4832-4969 Sentence denotes The study identified eight drugs including raloxifene, sildenafil, cefepime, cefpirome, imatinib, ponatinib, abemaciclib and entrectinib.
T32 4970-5152 Sentence denotes Subsequently, the eight compounds were purchased and tested for their capacity to displace a reference ligand in any of the two bradykinin receptors B1 and B2, respectively at 20 µM.
T33 5153-5331 Sentence denotes Three of the compounds including raloxifene, sildenafil and cefepime displaced the reference ligand from B2, which represents a 40% success rate as found in similar studies [29].
T34 5332-5424 Sentence denotes However, none of the compounds exhibited affinity for the B1 receptor at this concentration.
T35 5425-5530 Sentence denotes This might be due to the steric hindrance that was not properly included in the virtual screening search.
T36 5531-5667 Sentence denotes Raloxifene, the most potent antagonist identified was further investigated in a functional study in vitro, exhibiting an IC50 of ~16 µM.
T37 5668-5761 Sentence denotes Moreover, the compound showed a weak partial agonist profile with a maximal activity of ~20%.
T38 5762-5927 Sentence denotes Ralixofene is a selective estrogen receptor modulator, exerting estrogen agonist action in some target tissues while acting as an estrogen antagonist in others [30].
T39 5928-6067 Sentence denotes The compound was approved a few years ago for the treatment of osteoporosis in postmenopausal women, as well as for cancer chemoprotection.
T40 6068-6254 Sentence denotes The discovery of a new pharmacodynamic profile of relixofene as a BK partial agonist opens an opportunity for its repurposing as a therapeutic agent for the treatment of severe Covid-19.
T41 6256-6258 Sentence denotes 2.
T42 6259-6281 Sentence denotes Results and Discussion
T43 6282-6503 Sentence denotes In order to have a preliminary insight into the affinity of ralixofene towards the bradykinin receptors, the compound was first tested at 20 µM for its capacity to displace a reference ligand from B1 and B2, respectively.
T44 6504-6608 Sentence denotes Specifically, desArg10-kallidin was used in the case of the B1 receptor and NPC 567 for the B2 receptor.
T45 6609-6805 Sentence denotes The results of this study indicate that at this concentration, ralixofene does not displace desArg10-kallidin at the B1 receptor, whereas the reference ligand at the B2 receptor is displaced ~54%.
T46 6806-6982 Sentence denotes We subsequently proceeded to carry out a study of the pharmacodynamic profile of the compound towards the BK B2 receptor in vitro, using a functional efficacy measurement [31].
T47 6983-7113 Sentence denotes Accordingly, we tested its capacity to antagonize BK on Chinese Hamster Ovary (CHO) cells stably transfected with the B2 receptor.
T48 7114-7471 Sentence denotes As can be seen in Figure 1a, raloxifene antagonizes BK with an IC50 ~16 µM with an apparent dissociation constant KB = 1.8 µM, computed using the modified Cheng Prusoff equation [32]:KB=IC501+AEC50 where, IC50 = 16 µM corresponds to raloxifene and EC50 = 2.4 pM corresponds to BK, and A = 0.03 nM is the concentration of BK that was used in the experiments.
T49 7472-7567 Sentence denotes We also tested its activity as an agonist on CHO cells stably transfected with the B2 receptor.
T50 7568-7734 Sentence denotes Present results also showed (Figure 1b) that the compound exhibited a partial agonistic profile, with a maximum effect of 20% of BK that was achieved at around 20 µM.
T51 7735-7817 Sentence denotes The antagonistic profile of the compound can be rationalized from docking studies.
T52 7818-8009 Sentence denotes Figure 2 shows the prospective bound conformation of raloxifene (orange) to the B2 receptor, superimposed with the conformation matching the query (yellow) from the virtual screening process.
T53 8010-8157 Sentence denotes Despite fulfilling the four pharmacophore points, the directionality of the hydrogen bond interactions (not included in the query) was not optimal.
T54 8158-8279 Sentence denotes However, in the docking process, the molecule was relaxed to satisfy new interactions that may improve its docking score.
T55 8280-8408 Sentence denotes As can be seen in Figure 2, from its initial position (yellow), the molecule tilts to get a few favorable interactions (orange).
T56 8409-8541 Sentence denotes Specifically, the phenoxyl moiety sacrifices a hydrogen bond interaction with Arg169 that is compensated with a new one with Asn104.
T57 8542-8689 Sentence denotes This in turn, permits the phenoxyl aromatic ring to get closer to Trp86 and the carbonyl group establishes a hydrogen bond interaction with Arg169.
T58 8690-8860 Sentence denotes On the other hand, some interactions are weakened, for example, the interaction of the bicycle moiety with Trp86 and the interaction of the pyridine nitrogen with Asp266.
T59 8861-8955 Sentence denotes Partial fulfilment of the pharmacophore may explain the low IC50 exhibited by raloxifene [26].
T60 8956-9142 Sentence denotes On the other hand, the fact that raloxifene does not bind to the B1 can only be explained by steric interactions, since most of the residues between the two receptors are conserved [27].
T61 9143-9355 Sentence denotes Inspection of the results of the docking study reveals that the pyridine nitrogen of raloxifene exhibits a hydrogen bond with Thr286 that cannot not be established in B1, since the position is occupied by Leu294.
T62 9356-9470 Sentence denotes Bradykinin signaling though the B2 receptor induces a proinflammatory action as well as a hypotensive effect [33].
T63 9471-9703 Sentence denotes The proinflammatory activity of BK involves the stimulation of cytokines, like IL-6 and IL-8, through the MAPK/AP-1 signaling axis [17,34], whereas the hypotensive effects are due to vasodilation and increased vascular permeability.
T64 9704-9893 Sentence denotes Due to the partial agonist profile of raloxifene, it is expected that the compound antagonizes the proinflammatory effects of BK, modulating the levels of diverse cytokines, including IL-6.
T65 9894-10241 Sentence denotes To support this hypothesis, in clinical trials aimed at understanding the effect of hormone replacement therapy for menopausal women on the risk of cardiovascular disease, in contrast to other compounds raloxifene did not exhibit a proinflammatory profile [35,36] that could, in part, be explained by its antagonistic effect on the BK B2 receptor.
T66 10242-10380 Sentence denotes The beneficial vascular effects of ralixofene could, in part, be explained by its partial agonist profile towards the BK B2 receptor [36].
T67 10381-10647 Sentence denotes Thus, in a study aimed at assessing the vasoprotective effects of the compound, rats treated with raloxifene showed an increased reduction of systolic blood pressure on administration of bradykinin, suggesting an enhanced bioavailability of NO in these animals [37].
T68 10648-10845 Sentence denotes Moreover, diverse experiments subsequently confirmed a role of raloxifene inducing endothelium-dependent relaxation as due to the upregulation of nitric-oxide synthase (eNOS) expression [38,39,40].
T69 10846-11038 Sentence denotes Although these results can be due to the partial agonist profile of raloxifene reported in this work [33], the effect can also be explained as due to the activation of the GPR30 receptor [41].
T70 11039-11121 Sentence denotes Accordingly, further experimental work is necessary to differentiate both effects.
T71 11122-11276 Sentence denotes Present results strongly support the need to perform a deeper investigation on the use of raloxifene as a therapeutic agent for the treatment of Covid-19.
T72 11277-11487 Sentence denotes As shown in the present communication, its pharmacological profile as a partial BK B2 agonist is expected to antagonize the inflammatory action of BK, shown to be upregulated in Covid-19 patients [12,13,14,15].
T73 11488-11660 Sentence denotes This, in turn, will produce a reduction of cytokine levels, including IL-6, which is the primary proinflammatory cytokine associated with the severity of the illness [5,6].
T74 11661-11806 Sentence denotes On the other hand, its partial agonism profile is expected to maintain a base level of BK, preserving its beneficial cardiovascular effects [42].
T75 11807-11977 Sentence denotes Despite the novel beneficial effects identified, caution should be paid due to the necessary higher dose required for the compound to be used for a novel therapeutic use.
T76 11978-12053 Sentence denotes The treatment of osteoporosis requires an oral dose between 30 to 150 mg/d.
T77 12054-12184 Sentence denotes Taking into account its poor bioavailability of 2%, a single dose of 100 mg gives a maximal concentration in plasma of ~2 nM [43].
T78 12185-12391 Sentence denotes Concentrations necessary for the therapeutical benefit of raloxifene towards the BK B2 receptor are expected to be, at most, around 100 times higher, which is within the therapeutic window of the drug [44].
T79 12392-12587 Sentence denotes The results of this work complement recent comments suggesting the use of raloxifene for the treatment of Covid-19 [45], based on its capacity to inhibit the IL-6/STAT3 signaling pathway [46,47].
T80 12588-12792 Sentence denotes In addition, the presumed antiviral profile of ralixofene against the SARS-Cov2 virus through its capability to block the two-pore channel TCP2 [48], as found for the ebola virus [49] has been underlined.
T81 12793-12972 Sentence denotes In conclusion, the pharmacodynamic profile exhibited by ralidoxene described in the present report represents a reasonable base to test the compound for the treatment of Covid-19.
T82 12973-13095 Sentence denotes Very recently a clinical trial in Italy has been approved to test ralixofene in COVID-19 patients with mild symptoms [50].
T83 13097-13099 Sentence denotes 3.
T84 13100-13121 Sentence denotes Materials and Methods
T85 13123-13127 Sentence denotes 3.1.
T86 13128-13149 Sentence denotes Computational Methods
T87 13150-13381 Sentence denotes Vitual screening on the DrugBank [23] was carried out using the Molecular Operating Environment (MOE) program [28], using four common points of the BK B1 and B2 pharmacophores described previously [25,26] that were used as a query.
T88 13382-13545 Sentence denotes Previously, a 3D DrugBank database containing the 3D structure, together with a set of conformations of each molecule, was constructed as explained elsewhere [24].
T89 13546-13677 Sentence denotes Virtual screening was carried out on the subset of molecules with molecular weight between 200 and 600 (a total of 1703 molecules).
T90 13678-13953 Sentence denotes Hits identified in the screening process were docked in the 3D models of the B1 and B2 bradykinin receptors, using a set of unique conformations resulting from thorough conformational searches for the diverse ligands studied and rank ordered by means of the MOE program [28].
T91 13955-13959 Sentence denotes 3.2.
T92 13960-13975 Sentence denotes In Vitro Assays
T93 13977-13983 Sentence denotes 3.2.1.
T94 13984-14010 Sentence denotes Ligand Displacement Assays
T95 14011-14177 Sentence denotes Raloxifene was tested for its ability to displace reference ligands on human recombinant bradykinin B1 or B2 receptors, respectively, expressed in CHO cells at 20 µM.
T96 14178-14386 Sentence denotes For this purpose, first saturation isotherms were obtained with reference ligands ([3H] des-Arg10-BK (0.35 nM) in the case of B1, and [3H]-bradykinin (0.2 nM) for B2) incubated for 60 min at room temperature.
T97 14387-14520 Sentence denotes Nonspecific binding was evaluated by adding a reference compound (desArg9[Leu8]-BK at 10 µM in the case of B1 and BK at 1 µM for B2).
T98 14521-14753 Sentence denotes Antagonism of raloxifene was measured as a percent inhibition of specific binding of [3H]-bradykinin as control, obtained in the presence of raloxifene at 20 µM and using as reference compounds desArg10-KD for B1 and NPC-567 for B2.
T99 14755-14761 Sentence denotes 3.2.2.
T100 14762-14788 Sentence denotes Functional Efficacy Assays
T101 14789-14919 Sentence denotes Measurementent of the efficacy of the compound on the B2 receptor was carried out following the protocol described elsewhere [31].
T102 14920-15102 Sentence denotes The method is based on measuring differences in intracellular Ca2+ concentrations produced in diverse conditions on CHO cells expressing recombinant human B2 receptor by fluorometry.
T103 15103-15223 Sentence denotes Agonism was measured through the capacity of the compound to increase Ca2+ concentration compared to BK (EC50 ~ 2.4 pM).
T104 15224-15331 Sentence denotes Antagonism was measured through the capacity to antagonize BK based on the reduction of Ca2+ concentration.
T105 15333-15335 Sentence denotes 4.
T106 15336-15347 Sentence denotes Conclusions
T107 15348-15472 Sentence denotes The aim of the present study was to characterize the pharmacological profile of raloxifene towards the bradykinin receptors.
T108 15473-15595 Sentence denotes The compound was identified as a micromolar ligand of the B2 receptor by virtual screening in a drug repurposing campaign.
T109 15596-15771 Sentence denotes Results showed that raloxifene is a weak partial agonist toward the B2 receptor, with a 19% efficacy compared to bradykinin, with an apparent dissociation constant KB ~ 21 µM.
T110 15772-15988 Sentence denotes The discovery of the bradykinin pharmacodynamics profile of raloxidene explains in part its observed vascular beneficial effects, although they could also be due to activation of the GPR30 receptor signaling pathway.
T111 15989-16071 Sentence denotes On the other hand, it acts as a bradykinin antagonist for B2 with an IC50 ~ 21 µM.
T112 16072-16250 Sentence denotes Based on the bradykinin hypothesis on the trigger of the cytokine storm in severe Covid-19 [12,13,14,15], these results suggest that the compound could be used for its treatment.
T113 16251-16506 Sentence denotes Present results support previous claims suggesting the use of raloxifene based on its capacity to inhibit the IL-6/STAT3 signaling pathway [45], and also for being a prospective antiviral agent for SARS-Cov2 as inhibitor of the two-pore channel TCP2 [48].
T114 16507-16631 Sentence denotes Very recently a clinical trial in Italy has been approved to test its efficacy in COVID-19 patients with mild symptoms [50].
T115 16633-16648 Sentence denotes Acknowledgments
T116 16649-16823 Sentence denotes The antagonist potency of the hits discovered in the in silico study were carried out by Cerep under contracts FR095-0020745 and FR095-0021802 and is gratefully acknowledged.
T117 16824-16947 Sentence denotes Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
T118 16949-16969 Sentence denotes Author Contributions
T119 16970-17077 Sentence denotes Conceptualization, J.J.P.; methodology, P.G.-G.; calculations and formal analysis, P.G.-G.; writing, J.J.P.
T120 17078-17154 Sentence denotes All authors have read and agreed to the published version of the manuscript.
T121 17156-17163 Sentence denotes Funding
T122 17164-17313 Sentence denotes The present study was funded by the Spanish Ministry of Science and Innovation through the Carlos III Institute of Health under contract COV20/00052.
T123 17315-17351 Sentence denotes Institutional Review Board Statement
T124 17352-17367 Sentence denotes Not applicable.
T125 17369-17395 Sentence denotes Informed Consent Statement
T126 17396-17411 Sentence denotes Not applicable.
T127 17413-17440 Sentence denotes Data Availability Statement
T128 17441-17531 Sentence denotes The data presented in this study are available upon request from the corresponding author.
T129 17533-17554 Sentence denotes Conflicts of Interest
T130 17555-17599 Sentence denotes The authors declare no conflict of interest.
T131 17601-17853 Sentence denotes Figure 1 Functional efficacy study of raloxifene on CHO cells stably transfected with the B2 receptor (N = 2). (a) Study of the agonistic effect of the compound compared to bradykinin; (b) study of the efficacy of the compound antagonizing bradykinin.
T132 17854-18045 Sentence denotes Figure 2 Superposition of the prospective bound conformation of raloxifene onto the B2 receptor (orange) and the matching structure of raloxifene (yellow) from the virtual screening process.
T133 18046-18148 Sentence denotes The 3D structure of the B2 receptor was constructed by homology modeling and reported previously [27].
T134 18149-18427 Sentence denotes Color spheres represent the pharmacophore points characteristic of the ligands binding to B2 receptor: dark blue represents a positive charge moiety; magenta a hydrogen bond accepting center; light blue a hydrogen bond donor/acceptor center; green an aromatic/lipophilic center.