Cryptosporidium is a widely distributed enteric parasite that has an increasingly appreciated pathogenic role, particularly in pediatric diarrhea. While cryptosporidiosis has likely affected humanity ...for millennia, its recent "emergence" is largely the result of discoveries made through major epidemiologic studies in the past decade. There is no vaccine, and the only approved medicine, nitazoxanide, has been shown to have efficacy limitations in several patient groups known to be at elevated risk of disease. In order to help frontline health workers, policymakers, and other stakeholders translate our current understanding of cryptosporidiosis into actionable guidance to address the disease, we sought to assess salient issues relating to clinical management of cryptosporidiosis drawing from a review of the literature and our own field-based practice. This exercise is meant to help inform health system strategies for improving access to current treatments, to highlight recent achievements and outstanding knowledge and clinical practice gaps, and to help guide research activities for new anti-Cryptosporidium therapies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The discovery and development of TB drugs has met limited success, with two new drugs approved over the last 40 years. Part of the difficulty resides in the lack of well-established in vitro or in ...vivo targets of potency and physicochemical and pharmacokinetic parameters. In an attempt to benchmark and compare such properties for anti-TB agents, we have experimentally determined and compiled these parameters for 36 anti-TB compounds, using standardized and centralized assays, thus ensuring direct comparability across drugs and drug classes.
Potency parameters included growth inhibition, cidal activity against growing and non-growing bacteria and activity against intracellular mycobacteria. Pharmacokinetic parameters included basic physicochemical properties, solubility, permeability and metabolic stability. We then attempted to establish correlations between physicochemical, in vitro and in vivo pharmacokinetic and pharmacodynamic indices to tentatively inform future drug discovery efforts.
Two-thirds of the compounds tested showed bactericidal and intramacrophage activity. Most compounds exhibited favourable solubility, permeability and metabolic stability in standard in vitro pharmacokinetic assays. An analysis of human pharmacokinetic parameters revealed associations between lipophilicity and volume of distribution, clearance, plasma protein binding and oral bioavailability. Not surprisingly, most compounds with favourable pharmacokinetic properties complied with Lipinski's rule of five.
However, most attempts to detect in vitro-in vivo correlations were unsuccessful, emphasizing the challenges of anti-TB drug discovery. The objective of this work is to provide a reference dataset for the TB drug discovery community with a focus on comparative in vitro potency and pharmacokinetics.
Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with ...particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.
Indole-2-carboxamides have been identified as a promising class of antituberculosis agents from phenotypic screening against mycobacteria. One of the hits, indole-2-carboxamide analog (1), had low ...micromolar potency against Mycobacterium tuberculosis (Mtb), high mouse liver microsomal clearance, and low aqueous solubility. Structure–activity relationship studies revealed that attaching alkyl groups to the cyclohexyl ring significantly improved Mtb activity but reduced solubility. Furthermore, chloro, fluoro, or cyano substitutions on the 4- and 6-positions of the indole ring as well as methyl substitution on the cyclohexyl ring significantly improved metabolic stability. 39 and 41, the lead candidates, displayed improved in vitro activity compared to most of the current standard TB drugs. The low aqueous solubility could not be mitigated because of the positive correlation of lipophilicity with Mtb potency. However, both compounds displayed favorable oral pharmacokinetic properties in rodents and demonstrated in vivo efficacy. Thus, indole-2-carboxamides represent a promising new class of antituberculosis agents.
Pyridone 1 was identified from a high-throughput cell-based phenotypic screen against Mycobacterium tuberculosis (Mtb) including multi-drug resistant tuberculosis (MDR-TB) as a novel anti-TB agent ...and subsequently optimized series using cell-based Mtb assay. Preliminary structure activity relationship on the isobutyl group with higher cycloalkyl groups at 6-position of pyridone ring has enabled us to significant improvement of potency against Mtb. The lead compound 30j, a dimethylcyclohexyl group on the 6-position of the pyridone, displayed desirable in vitro potency against both drug sensitive and multi-drug resistant TB clinical isolates. In addition, 30j displayed favorable oral pharmacokinetic properties and demonstrated in vivo efficacy in mouse model. These results emphasize the importance of 4-hydroxy-2-pyridones as a new chemotype and further optimization of properties to treat MDR-TB.
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•Hit pyridone 1 inhibited growth of Mycobacterium tuberculosis (Mtb) including multi-drug resistant tuberculosis.•SAR on 6-position of pyridone 1 significantly improved potency and lead compound 30j displayed 50 nM activity against Mtb.•Lead 30j showed favorable oral PK and demonstrated in vivo efficacy in mouse model.•Mechanism of action studies indicated thatpyridones are direct inhibitors of InhA.
The bicyclic 4‐nitroimidazoles PA‐824 and OPC‐67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro‐drugs ...that are reductively activated by a deazaflavin (F420) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA‐824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo‐oxazoles bearing only short alkyl substituents at the C‐7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA‐824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA‐824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover‐independent assessment of affinity. Our results indicate that (R)‐PA‐824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C‐3 of the imidazole ring orienting in a non‐productive position with respect to the incoming hydride from F420. The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.
PA‐824, a bicyclic‐nitroimidazole in clinical development against tuberculosis, is bioreductively activated by a deazaflavin (F420) dependent nitroreductase (Ddn). Substrate and stereospecificity of Ddn is directed by the presence of a long hydrophobic tail. (R)‐PA‐824 binds Ddn with the same affinity as the active (S)‐PA‐824, despite not being turned over. Docking studies suggest that (R)‐PA‐824 likely binds non‐productively to the enzyme.
Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium ...tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F420 deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F420 and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.
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► Crystal structures of Ddn, the reductase for antitubercular drug PA-824 ► Active site residues and F420-binding mode described ► The N terminus is important for PA-824 binding but could not be fully characterized ► An active homolog from Nocardia farcinica was identified and studied
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Tuberculosis poses a major global health problem and multi-drug resistant strains are increasingly prevalent. Hence there is an urgent need to discover new TB drugs. Cell based ...phenotypic screening represents a powerful approach to identify anti-mycobacterial compounds and elucidate novel targets. Three high throughput phenotypic screens were performed at NITD against mycobacterium. Hits were identified and chemical series selected for optimisation. This produced compounds with good in vitro anti-mycobacterial activity and pharmacokinetic properties. Some compounds displayed oral activity in mouse efficacy models of TB. Herein, we review the TB discovery efforts at NITD and share experiences in optimisation of phenotypic hits, describing challenges encountered and lessons learned. We also offer perspectives to facilitate future selection and advancement of phenotypic hits.
PA-824 is a bicyclic 4-nitroimidazole, currently in phase II clinical trials for the treatment of tuberculosis. Dose fractionation pharmacokinetic-pharmacodynamic studies in mice indicated that the ...driver of PA-824 in vivo efficacy is the time during which the free drug concentrations in plasma are above the MIC (fT>MIC). In this study, a panel of closely related potent bicyclic 4-nitroimidazoles was profiled in both in vivo PK and efficacy studies. In an established murine TB model, the efficacy of diverse nitroimidazole analogs ranged between 0.5 and 2.3 log CFU reduction compared to untreated controls. Further, a retrospective analysis was performed for a set of seven nitroimidazole analogs to identify the PK parameters that correlate with in vivo efficacy. Our findings show that the in vivo efficacy of bicyclic 4-nitroimidazoles correlated better with lung PK than with plasma PK. Further, nitroimidazole analogs with moderate-to-high volume of distribution and Lung to plasma ratios of >2 showed good efficacy. Among all the PK-PD indices, total lung T>MIC correlated the best with in vivo efficacy (rs = 0.88) followed by lung Cmax/MIC and AUC/MIC. Thus, lung drug distribution studies could potentially be exploited to guide the selection of compounds for efficacy studies, thereby accelerating the drug discovery efforts in finding new nitroimidazole analogs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The GEMS study estimated about 7.5 million cases of Cryptosporidium infection occur every year within this population in Africa and Asia resulting in over 200 000 Cryptosporidium-attributable deaths ...due to moderate-to-severe diarrhoea, with an excess of 59 000 deaths compared with children with similar symptoms that were Cryptosporidium negative.2 Cryptosporidium infection in these malnourished children is also significantly associated with debilitating stunted growth contributing to excess mortality.3–6 This Cryptosporidium-associated stunting and wasting leads to poor physical and neurological health with poor childhood development, resulting in a lasting effect on population health in LMICs.5 This burden falls disproportionately on children in sub-Saharan Africa, but also in South America and Asia (figure 1). Multiple such studies have been conducted with Cryptosporidium in the past and were found to be safe34–36 and the model has recently be updated.37 We support a clinical trial plan proposed in which the proof of concept (phase 2a study) is conducted with volunteers intentionally infected with C. parvum, followed by phase 2b and 3 studies in children in endemic areas.37 Since malaria and Cryptosporidium belong to the same phylum Apicomplexa, they share some conserved drug targets, and thus there is a synergy possibility in research and development of malaria and Cryptosporidium therapeutics. There are also possibilities for synergy with the animal health market, particularly for dairy cattle, where in some areas nearly 100% of newborn calves acquire C. parvum infection, and Cryptosporidium infection has been shown to lead to lasting weight loss and reduced milk production.39–41 A challenge to be addressed is the clinical usage of an anti-Cryptosporidium drug. There is current compelling evidence of unmet therapeutic need for enteric cryptosporidiosis found in three patient groups: (1) young children aged 0–24 months in LMICs; (2) malnourished children under age 5 and (3) immunosuppressed individuals of any age.42 43 A recent publication outlines an effective therapeutic could be used to reduce the large burden of Cryptosporidium in LMICs (table 2).42 Cryptosporidium therapy could be used syndromically, for instance in children less than 2 years old with moderate-to-severe diarrhoea, probably combined with an antibacterial to cover the major treatable causes, E. coli, Shigella spp. and C. jejuni.
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