Drugs Repurposing for Multi-Drug Resistant Bacterial Infections

Different institutions recognized that antimicrobial resistance is a global health threat that has compounded by the reduction in the discovery and development of new antimicrobial agents. Therefore, the development of new antimicrobial therapeutic strategies requires immediate attention to avoid the 10 million deaths predicted to occur by 2050 as a result of multidrug-resistant (MDR) bacteria. Despite the great interest in the development of repurposing drugs, only few repurposing drugs are under clinical development against Gram-negative critical-priority pathogens. In this chapter, we aim: (i) to discuss the therapeutic potential of the repurposing drugs for treating MDR bacterial infections, (ii) to summarize their mechanism of action, and (iii) to provide an overview for their preclinical and clinical development against these critical-priority pathogens

Synergistic Activity of Niclosamide in Combination With Colistin Against Colistin-Susceptible and Colistin-Resistant Acinetobacter baumannii and Klebsiella pneumoniae

Colistin is among the few antibiotics effective against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant A. baumannii and K. pneumoniae strains have emerged. Therefore, combination therapies, between colistin and other old drugs, restoring the activity of colistin are required. The main objective of this study was to analyse the activity of niclosamide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) A. baumannii and K. pneumoniae. The MIC were determined by microdilution assay and the time-kill curves were performed. The zeta potential of Col-S and Col-R of A. baumannii and K. pneumoniae in presence of niclosamide was assessed. Niclosamide in combination with colistin showed improved activity against Col-S and Col-R A. baumannii and K. pneumoniae. Time-killing curves showed synergic activity between niclosamide and colistin against Col-S and Col-R A. baumannii and K. pneumoniae, especially when niclosamide or colistin was added for second time at 4 h of the 24 h killing curve. Col-R A. baumannii and K. pneumoniae in presence of niclosamide exhibited a greater negative charge (−34.95 ± 0.35 mV and −38.85 ± 0.92 mV; P < 0.05) than Col-R A. baumannii and K. pneumoniae in absence of niclosamide (−26.85 ± 3.65 mV and −35.27 ± 0.72 mV). These data suggest that niclosamide might be combined with colistin, being a potential alternative for treatment of Col-R Gram-negative bacilli infections.Instituto de Salud Carlos IIIProyectos de Investigacion en Salud PI16/01378Ministerio de Economía y Competitividad CP15/0135

Table2_Expanding the phenotype of THRB: a range of macular dystrophies as the major clinical manifestations in patients with a dominant splicing variant.PDF

Supplementary Table 2 Different THRB in vivo and in vitro knock-out and knockdown models and its phenotypic effect.Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms.Peer reviewe

Expanding the phenotype of THRB: a range of macular dystrophies as the major clinical manifestations in patients with a dominant splicing variant

© 2023 Fernández-Suárez, González-del Pozo, García-Núñez, Méndez-Vidal, Martín-Sánchez, Mejías-Carrasco, Ramos-Jiménez, Morillo-Sánchez, Rodríguez-de la Rúa, Borrego and Antiñolo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms.This work was supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation, Spain and co-funded by ERDF (“A way to make Europe”) [PI21-00244]; The strategic plan for the Precision Medicine Infrastructure associated with Science and Technology - IMPaCT [IMP-0009], Regional Ministry of Health and Families of the Autonomous Government of Andalusia [PEER-0501-2019] and the Foundation Isabel Gemio/Foundation Cajasol [FGEMIO-2019-01]. EF-S is supported by fellowship FI19/00091 from ISCIII (ESF, “Investing in your future”). MM-S [RH-0049-2021] are supported by a fellowship funded by the Regional Ministry of Health and Families of the Autonomous Government of Andalusia.Peer reviewe

Image1_Expanding the phenotype of THRB: a range of macular dystrophies as the major clinical manifestations in patients with a dominant splicing variant.PDF

Supplementary figure S1. Schematic representation of the hypothetical consequences of the THRB variant. In silico prediction of the effect of the spliceogenic variant identified in TRβ1 isoform (NM_001354712.2: c.283+1G>A) showed two scenarios. (A), The skipping of exon 5 disrupts the A/B domain, leaving a protein of 374 amino acids with a secondary structure different from the wild-type protein. The DNAbinding domain (DBD), hinge domain and ligand-binding domain (LBD) are not affected. (B), The skipping of exons 5 and 6 results in a premature codon stop that produces only a small peptide. The 3D models of the proteins were generated using the IntFOLD and PEP-FOLD4 online servers.Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms.Peer reviewe

O31 Integrative analysis reveals a molecular stratification of systemic autoimmune diseases

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Repurposing of the tamoxifen metabolites in combination with tigecycline against Gram-negative bacteria

Motivation: Emerging of multidrug-resistant (MDR) bacteria represent a matter of grave urgency and a problem for public health. Due to the emergence of resistance new strategic antimicrobial therapeutic approaches are proposed, such as drug repurposing. Tamoxifen was previously reported to present efficacy against MDR Acinetobacter baumannii and Escherichia coli [1]. The objetive of this project was to study in vitro the activity of the three major metabolites of tamoxifen (MET): N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen, in combination with tigecycline against colistin-susceptible (COL-S) and colistin-resistant (COL-R) A. baumannii and E. coli. Methods: A colection of Gram-negative bacteria [8 COL-R and 1 COL-S A. baumannii, 17 COL-R and 1 COL-S E. coli] was used [2]. All strains were grown in Mueller-Hinton Broth (MHB) at 37ºC. Minimal Inhibitory Concentration (MIC) was determined for all strains by using microdilution assay. In order to determine the synergy between a mix of the three MET and tigecycline checkerboard and time-kill curves assays were performed. Results: Tigecycline MIC range was 4-8 mg/L for all COL-R A. baumannii strains, 0.5 mg/L for COL-S A. baumannii strain and 0.125-1 mg/L for both COL-R and COL-S E. coli strains. Checkerboard analyses showed partial synergism for combination tigecycline and MET against COL-R ans COL-S A. baumannii and E. coli strains. Time-kill curves confirmed synergetic effect and inhibited partially and completely the regrowth of COL-R E. coli and A. baumannii strains, respectively. Conclusions: Tamoxifen metabolites in combination with tigecycline showed in vitro synergetic effect against COL-R A. baumannii and E. coli strains, representing a potential new alternative for treatment of infections caused by MDR A. baumannii and E. coli

Efficacy of lysophosphatidylcholine as direct treatment in combination with colistin against acinetobacter baumannii in murine severe infections models

The stimulation of the immune response to prevent the progression of an infection may be an adjuvant to antimicrobial treatment. Here, we aimed to evaluate the efficacy of lysophosphatidylcholine (LPC) treatment in combination with colistin in murine experimental models of severe infections by Acinetobacter baumannii. We used the A. baumannii Ab9 strain, susceptible to colistin and most of the antibiotics used in clinical settings, and the A. baumannii Ab186 strain, susceptible to colistin but presenting a multidrug-resistant (MDR) pattern. The therapeutic efficacies of one and two LPC doses (25 mg/kg/d) and colistin (20 mg/kg/8 h), alone or in combination, were assessed against Ab9 and Ab186 in murine peritoneal sepsis and pneumonia models. One and two LPC doses combined with colistin and colistin monotherapy enhanced Ab9 and Ab186 clearance from spleen, lungs and blood and reduced mice mortality compared with those of the non-treated mice group in both experimental models. Moreover, one and two LPC doses reduced the bacterial concentration in tissues and blood in both models and increased mice survival in the peritoneal sepsis model for both strains compared with those of the colistin monotherapy group. LPC used as an adjuvant of colistin treatment may be helpful to reduce the severity and the resolution of the MDR A. baumannii infection

Potential Tamoxifen Repurposing to Combat Infections by Multidrug-Resistant Gram-Negative Bacilli

The development of new strategic therapies for multidrug-resistant bacteria, like the use of non-antimicrobial approaches and/or drugs repurposed to be used as monotherapies or in combination with clinically relevant antibiotics, has become urgent. A therapeutic alternative for infections by multidrug-resistant Gram-negative bacilli (MDR-GNB) is immune system modulation to improve the infection clearance. We showed that immunocompetent mice pretreated with tamoxifen at 80 mg/kg/d for three days and infected with Acinetobacter baumannii, Pseudomonas aeruginosa, or Escherichia coli in peritoneal sepsis models showed reduced release of the monocyte chemotactic protein-1 (MCP-1) and its signaling pathway interleukin-18 (IL-18), and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). This reduction of MCP-1 induced the reduction of migration of inflammatory monocytes and neutrophils from the bone marrow to the blood. Indeed, pretreatment with tamoxifen in murine peritoneal sepsis models reduced the bacterial load in tissues and blood, and increased mice survival from 0% to 60-100%. Together, these data show that tamoxifen presents therapeutic efficacy against MDR A. baumannii, P. aeruginosa, and E. coli in experimental models of infection and may be a new candidate to be repurposed as a treatment for GNB infections.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (grants CP15/00132, PI16/01378 and PI19/01453) and by Plan Nacional de I+D+i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Coopera tiva, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014–2020. Younes Smani is supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/00132).Ye