RESUMEN
The World Health Organization (WHO) has warned that our current arsenal of antibiotics is not innovative enough to face impending infectious diseases, especially those caused by multidrug-resistant Gram-negative pathogens. Although the current preclinical pipeline is well stocked with novel candidates, the last U.S. Food and Drug Administration (FDA)-approved antibiotic with a novel mechanism of action against Gram-negative bacteria was discovered nearly 60 years ago. Of all the antibiotic candidates that initiated investigational new drug (IND) applications in the 2000s, 17% earned FDA approval within 12 years, while an overwhelming 62% were discontinued in that time frame. These "leaks" in the clinical pipeline, where compounds with clinical potential are abandoned during clinical development, indicate that scientific innovations are not reaching the clinic and providing benefits to patients. This is true for not only novel candidates but also candidates from existing antibiotic classes with clinically validated targets. By identifying the sources of the leaks in the clinical pipeline, future developmental efforts can be directed toward strategies that are more likely to flow into clinical use. In this review, we conduct a detailed failure analysis of clinical candidates with Gram-negative activity that have fallen out of the clinical pipeline over the past decade. Although limited by incomplete data disclosure from companies engaging in antibiotic development, we attempt to distill the developmental challenges faced by each discontinued candidate. It is our hope that this insight can help de-risk antibiotic development and bring new, effective antibiotics to the clinic.
Asunto(s)
Antibacterianos , Infecciones por Bacterias Gramnegativas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5'-ended strand and its slower RecB helicase on the 3'-ended strand. At Chi hotspots (5' GCTGGTGG 3'), RecB's nuclease cuts the 3'-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB's ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.
Asunto(s)
Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Exodesoxirribonucleasa V/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Bencimidazoles/química , Sitios de Unión , Inhibidores Enzimáticos/química , Exodesoxirribonucleasa V/química , Exodesoxirribonucleasa V/genética , Exodesoxirribonucleasa V/metabolismo , MutaciónRESUMEN
New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against Pseudomonas aeruginosa We report here the in vitro antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in phase 1 clinical trials. In addition, we describe the profiles of three additional LpxC inhibitors that were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Catálisis/efectos de los fármacos , Humanos , Pseudomonas aeruginosa/metabolismoRESUMEN
UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30â mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Diinos/farmacología , Inhibidores Enzimáticos/farmacología , Corazón/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/farmacocinética , Antibacterianos/toxicidad , Proteínas Bacterianas/antagonistas & inhibidores , Cardiotoxicidad , Diinos/síntesis química , Diinos/farmacocinética , Diinos/toxicidad , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/toxicidad , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacocinética , Ácidos Hidroxámicos/toxicidad , Masculino , Estructura Molecular , Profármacos/síntesis química , Profármacos/farmacocinética , Profármacos/farmacología , Profármacos/toxicidad , Pseudomonas aeruginosa/efectos de los fármacos , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10-8 and 10-9. However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to >128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Membrana Externa Bacteriana/efectos de los fármacos , Membrana Externa Bacteriana/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Fenómenos Químicos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/enzimología , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Modelos Químicos , Estructura Molecular , Mutación , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genéticaRESUMEN
The efficacy of plazomicin for pneumonic plague was evaluated in a non-human primate model. African Green monkeys challenged with a lethal aerosol of Yersinia pestis [median (range) of 98 (15-331) LD50s] received placebo (n=12) or 'humanized' dose regimens (6.25, 12.5 or 25mg/kg every 24h) of plazomicin (n=52) after the onset of fever for a duration of 5 or 10days. All animals treated with placebo died, while 36 plazomicin-treated animals survived through study end. The majority (33/36) were either in the 10-day (high-/mid-/low-dose) or 5-day high-dose groups. The findings suggest an exposure range of plazomicin for treatment of pneumonic/bacteremic Y. pestis infection in humans.
Asunto(s)
Modelos Animales de Enfermedad , Peste/tratamiento farmacológico , Sisomicina/análogos & derivados , Animales , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Conformación Molecular , Sisomicina/química , Sisomicina/uso terapéuticoRESUMEN
As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252-14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of ß-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site.
RESUMEN
The growing threat of untreatable bacterial infections has refocused efforts to identify new antibiotics, especially those acting by novel mechanisms. While the inhibition of pathogen proteases has proven to be a successful strategy for drug development, such inhibitors are often limited by toxicity due to their promiscuous inhibition of homologous and mechanistically related human enzymes. Unlike many protease inhibitors, inhibitors of the essential type I bacterial signal peptidase (SPase) may be more specific and thus less toxic due to the enzyme's unique structure and catalytic mechanism. Recently, the arylomycins and related lipoglycopeptide natural products were isolated and shown to inhibit SPase. The core structure of the arylomycins and lipoglycopeptides consists of a biaryl-linked, N-methylated peptide macrocycle attached to a lipopeptide tail, and in the case of the lipoglycopeptides, a deoxymannose moiety. Herein, we report the first total synthesis of a member of this group of antibiotics, arylomycin A2. The synthesis relies on Suzuki-Miyaura-mediated biaryl coupling, which model studies suggested would be more efficient than a lactamization-based route. Biological studies demonstrate that these compounds are promising antibiotics, especially against Gram-positive pathogens, with activity against S. epidermidis that equals that of the currently prescribed antibiotics. Structural and biological studies suggest that both N-methylation and lipidation may contribute to antibiotic activity, whereas glycosylation appears to be generally less critical. Thus, these studies help identify the determinants of the biological activity of arylomycin A2 and should aid in the design of analogs to further explore and develop this novel class of antibiotic.
Asunto(s)
Oligopéptidos/química , Oligopéptidos/farmacología , Catálisis , Humanos , Enlace de Hidrógeno , Estructura MolecularRESUMEN
Mutation is the driving force behind many processes linked to human disease, including cancer, aging, and the evolution of drug resistance. Mutations have traditionally been considered the inevitable consequence of replicating large genomes with polymerases of finite fidelity. Observations over the past several decades, however, have led to a new perspective on the process of mutagenesis. It has become clear that, under some circumstances, mutagenesis is a regulated process that requires the induction of pro-mutagenic enzymes and that, at least in bacteria, this induction may facilitate evolution. Herein, we review what is known about induced mutagenesis in bacteria as well as evidence that it contributes to the evolution of antibiotic resistance. Finally, we discuss the possibility that components of induced mutation pathways might be targeted for inhibition as a novel therapeutic strategy to prevent the evolution of antibiotic resistance.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Evolución Molecular , Mutación , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Humanos , Modelos Biológicos , MutagénesisRESUMEN
Staphylococcus aureus infections can be difficult to treat due to both multidrug resistance and the organism's remarkable ability to persist in the host. Persistence and the evolution of resistance may be related to several complex regulatory networks, such as the SOS response, which modifies transcription in response to environmental stress. To understand how S. aureus persists during antibiotic therapy and eventually emerges resistant, we characterized its global transcriptional response to ciprofloxacin. We found that ciprofloxacin induces prophage mobilization as well as significant alterations in metabolism, most notably the up-regulation of the tricarboxylic acid cycle. In addition, we found that ciprofloxacin induces the SOS response, which we show, by comparison of a wild-type strain and a non-SOS-inducible lexA mutant strain, includes the derepression of 16 genes. While the SOS response of S. aureus is much more limited than those of Escherichia coli and Bacillus subtilis, it is similar to that of Pseudomonas aeruginosa and includes RecA, LexA, several hypothetical proteins, and a likely error-prone Y family polymerase whose homologs in other bacteria are required for induced mutation. We also examined induced mutation and found that either the inability to derepress the SOS response or the lack of the LexA-regulated polymerase renders S. aureus unable to evolve antibiotic resistance in vitro in response to UV damage. The data suggest that up-regulation of the tricarboxylic acid cycle and induced mutation facilitate S. aureus persistence and evolution of resistance during antibiotic therapy.
Asunto(s)
Ciprofloxacina/farmacología , Respuesta SOS en Genética/fisiología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ciclo del Ácido Cítrico/genética , Ciclo del Ácido Cítrico/fisiología , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Respuesta SOS en Genética/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus/efectos de la radiación , Rayos UltravioletaRESUMEN
Pseudomonas aeruginosa infections can be virtually impossible to eradicate, and the evolution of resistance during antibiotic therapy is a significant concern. In this study, we use DNA microarrays to characterize the global transcriptional response of P. aeruginosa to clinical-like doses of the antibiotic ciprofloxacin and also to determine the component that is regulated by LexA cleavage and the SOS response. We find that genes involved in virtually every facet of metabolism are down-regulated in response to ciprofloxacin. The LexA-controlled SOS regulon identified by microarray analysis includes only 15 genes but does include several genes that encode proteins involved in recombination and replication, including two inducible polymerases known to play a role in mutation and the evolution of antibiotic resistance in other organisms. The data suggest that the inhibition of LexA cleavage during therapy might help combat this pathogen by decreasing its ability to adapt and evolve resistance.
Asunto(s)
Antibacterianos/farmacología , Ciprofloxacina/farmacología , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/genética , Respuesta SOS en Genética/genética , Proteínas Bacterianas/genética , ADN Polimerasa Dirigida por ADN/genética , Perfilación de la Expresión Génica , Genes Bacterianos , Análisis de Secuencia por Matrices de Oligonucleótidos , Pseudomonas aeruginosa/efectos de los fármacos , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Recombinasas/genética , Regulón , Serina Endopeptidasas/genéticaAsunto(s)
Antibacterianos/farmacología , ADN/farmacocinética , Evolución Molecular , Genes Bacterianos , Streptococcus pneumoniae/efectos de los fármacos , Cromosomas Bacterianos/genética , Farmacorresistencia Microbiana , Modelos Genéticos , Recombinación Genética , Streptococcus pneumoniae/genética , Transformación GenéticaRESUMEN
The emergence of drug-resistant bacteria poses a serious threat to human health. Bacteria often acquire resistance from a mutation of chromosomal genes during therapy. We have recently shown that the evolution of resistance to ciprofloxacin in vivo and in vitro requires the induction of a mutation that is mediated by the cleavage of the SOS repressor LexA and the associated derepression of three specialized DNA polymerases (polymerase II [Pol II], Pol IV, and Pol V). These results led us to suggest that it may be possible to design drugs to inhibit these proteins and that such drugs might be coadministered with antibiotics to prevent mutation and the evolution of resistance. For the approach to be feasible, there must not be any mechanisms through which bacteria can induce mutations and acquire antibiotic resistance that are independent of LexA and its repressed polymerases. Perhaps the most commonly cited mechanism to elevate bacterial mutation rates is the inactivation of methyl-directed mismatch repair (MMR). However, it is unclear whether this represents a LexA-independent mechanism or if the mutations that arise in MMR-deficient hypermutator strains are also dependent on LexA cleavage and polymerase derepression. In this work, we show that LexA cleavage and polymerase derepression are required for the evolution of clinically significant resistance in MMR-defective Escherichia coli. Thus, drugs that inhibit the proteins responsible for induced mutations are expected to efficiently prevent the evolution of resistance, even in MMR-deficient hypermutator strains.
Asunto(s)
Ciprofloxacina/farmacología , Replicación del ADN/efectos de los fármacos , Farmacorresistencia Bacteriana/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/efectos de los fármacos , Respuesta SOS en Genética/fisiología , Proteínas Bacterianas , Disparidad de Par Base/genética , Reparación del ADN/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Mutación , Serina EndopeptidasasRESUMEN
The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.