RESUMEN
Tuberculosis (TB) is one of the most significant world health problems, responsible for 1.5 M deaths in 2020, and yet, current treatments rely largely on 40 year old paradigms. Although the rifamycins (RIFs), best exemplified by the drug rifampin (RMP), represent a well-studied and therapeutically effective chemotype that targets the bacterial RNA polymerase (RNAP), these agents still suffer from serious drawbacks including the following: 3-9 month treatment times; cytochrome P450 (Cyp450) induction [particularly problematic for human immunodeficiency virus-Mycobacterium tuberculosis (MTB) co-infection]; and the existence of RIF-resistant (RIFR) MTB strains. We have utilized a structure-based drug design approach to synthesize and test 15 benzoxazinorifamycins (bxRIFs), congeners of the clinical candidate rifalazil, to minimize human pregnane X receptor (hPXR) activation while improving potency against MTB. We have determined the compounds' activation of the hPXR [responsible for inducing Cyp450 3A4 (CYP3A4)]. Compound IC50s have been determined against the wild-type and the most prevalent RIFR (ß-S450L) mutant MTB RNAPs. We have also determined their bactericidal activity against "normal" replicating MTB and a model for non-replicating, persister MTB. We have identified a minimal substitution and have probed larger substitutions that exhibit negligible hPXR activation (1.2-fold over the dimethyl sulfoxide control), many of which are 5- to 10-fold more potent against RNAPs and MTB than RMP. Importantly, we have analogues that are essentially equipotent against replicating MTB and non-replicating persister MTB, a property that is correlated with faster kill rates and may lead to shorter treatment durations. This work provides a proof of principle that the ansamycin core remains an attractive and effective scaffold for novel and dramatically improved RIFs.
Asunto(s)
Infecciones por VIH , Rifamicinas , Tuberculosis , Adulto , Infecciones por VIH/tratamiento farmacológico , Humanos , Receptor X de Pregnano , Rifampin/farmacología , Rifampin/uso terapéutico , Rifamicinas/farmacología , Rifamicinas/uso terapéutico , Tuberculosis/tratamiento farmacológicoRESUMEN
Rifampin (RMP), a very potent inhibitor of the Mycobacterium tuberculosis (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIFR) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIFR) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue (27a) that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (â¼30-fold > RMP) activity against RIFR MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, 27a has a 7 day MBC that is â¼10-fold more potent than RMP. These results suggest that 27a may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of â¼2 g of 27a at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies.
Asunto(s)
Mycobacterium tuberculosis , Rifamicinas , Tuberculosis , Animales , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Bacteriana , Ratones , Rifampin/farmacología , Rifamicinas/farmacología , Tuberculosis/tratamiento farmacológicoRESUMEN
There remain no approved therapies for rare but devastating neuronopathic glyocosphingolipid storage diseases, such as Sandhoff, Tay-Sachs, and Gaucher disease type 3. We previously reported initial optimization of the scaffold of eliglustat, an approved therapy for the peripheral symptoms of Gaucher disease type 1, to afford 2, which effected modest reductions in brain glucosylceramide (GlcCer) in normal mice at 60 mg/kg. The relatively poor pharmacokinetic properties and high Pgp-mediated efflux of 2 prompted further optimization of the scaffold. With a general objective of reducing topological polar surface area, and guided by multiple metabolite identification studies, we were successful at identifying 17 (CCG-222628), which achieves remarkably greater brain exposure in mice than 2. After demonstrating an over 60-fold improvement in potency over 2 at reducing brain GlcCer in normal mice, we compared 17 with Sanofi clinical candidate venglustat (Genz-682452) in the CBE mouse model of Gaucher disease type 3. At doses of 10 mg/kg, 17 and venglustat effected comparable reductions in both brain GlcCer and glucosylsphingosine. Importantly, 17 achieved these equivalent pharmacodynamic effects at significantly lower brain exposure than venglustat.
Asunto(s)
Enfermedad de Gaucher , Animales , Inhibidores Enzimáticos/farmacología , Enfermedad de Gaucher/tratamiento farmacológico , Glucosiltransferasas , Ratones , Pirrolidinas/farmacologíaRESUMEN
Clofazimine is a weakly basic, Food and Drug Administration-approved antibiotic recommended by the World Health Organization to treat leprosy and multi-drug-resistant tuberculosis. Upon prolonged treatment, clofazimine extensively bioaccumulates and precipitates throughout the organism, forming crystal-like drug inclusions (CLDIs). Due to the drug's red color, it is widely believed that clofazimine bioaccumulation results in skin pigmentation, its most common side effect. To test whether clofazimine-induced skin pigmentation is due to CLDI formation, we synthesized a closely related clofazimine analog that does not precipitate under physiological pH and chloride conditions that are required for CLDI formation. Despite the absence of detectable CLDIs in mice, administration of this analog still led to significant skin pigmentation. In clofazimine-treated mice, skin cryosections revealed no evidence of CLDIs when analyzed with a microscopic imaging system specifically designed for detecting clofazimine aggregates. Rather, the reflectance spectra of the skin revealed a signal corresponding to the soluble, free base form of the drug. Consistent with the low concentrations of clofazimine in the skin, these results suggest that clofazimine-induced skin pigmentation is not due to clofazimine precipitation and CLDI formation, but rather to the partitioning of the circulating, free base form of the drug into subcutaneous fat.
Asunto(s)
Clofazimina/toxicidad , Pigmentación de la Piel/efectos de los fármacos , Animales , Clofazimina/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células RAW 264.7RESUMEN
We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.
Asunto(s)
Ácidos Nipecóticos/farmacocinética , Ácidos Nipecóticos/uso terapéutico , Factor Rho/antagonistas & inhibidores , Esclerodermia Sistémica/tratamiento farmacológico , Piel/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Administración Oral , Animales , Modelos Animales de Enfermedad , Fibrosis , Células HEK293 , Humanos , Ratones , Ácidos Nipecóticos/administración & dosificación , Ácidos Nipecóticos/química , Factor Rho/metabolismo , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Elemento de Respuesta al Suero/efectos de los fármacos , Piel/metabolismo , Piel/patología , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismoRESUMEN
Doxorubicin and Cisplatin are the frontline therapeutics for treatment of the triple negative breast cancers (TNBCs). Emergence of drug-resistance often contributes to failure of drugs and poor prognosis, and thus necessitates development of new and improved modalities to treat TNBCs. We generated and characterized chemotherapy-resistant TNBC cells following their culture in chronic presence of Doxorubicin or Cisplatin, and tested whether their viabilities were inhibited by a novel class of CARP- 1 functional mimetic (CFM) compounds. Analogs of parent compound CFM-4 were obtained through structure-activity based medicinal chemistry studies. CFM-4.16, a novel analog of CFM-4, caused superior inhibition of viability of TNBC cells when used in combination with doxorubicin. Doxorubicin and cisplatin inhibited viabilities of parental cells with GI50 dose of 0.02-0.1 µM and 1.65 µM, respectively. The GI50 dose of doxorubicin for doxorubicin-resistant TNBC cells was ≥ 10.0 µM. For Cisplatin-resistant cells, the GI50 dose of Cisplatin was ≥ 6-15.0 µM for MDA-MB-468 sublines and ≥ 150.0 µM for MDA-MB-231 sublines. CFM-4.16 inhibited viability of chemotherapy-resistant TNBC cells, in part by inhibiting oncogenic cMet activation and expression, stimulating CARP-1 expression, caspase-8 cleavage and apoptosis. CFM-4.16 pretreatment enhanced anti-TNBC efficacies of inhibitors of cMET (Tevatinib) or cSrc (Dasatinib). CFM-4.16 suppressed growth of resistant TNBC cells in soft agar as well as in three-dimensional suspension cultures derived from enriched, stem-like cells. Finally, a nanolipid formulation of CFM-4.16 in combination with doxorubicin had superior efficacy in inhibiting TNBC xenograft growth. Our findings collectively demonstrate therapeutic potential of CFM-4.16 for parental and drug-resistant TNBCs.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Mimetismo Biológico , Proteínas de Ciclo Celular/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Tiadiazoles/química , Tiadiazoles/farmacología , Neoplasias de la Mama Triple Negativas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clofazimine is an orally administered, FDA-approved drug that massively bioaccumulates in macrophages, forming membrane-bound intracellular structures possessing nanoscale supramolecular features. Here, a library of phenazine compounds derived from clofazimine was synthesized and tested for their ability to accumulate and form ordered molecular aggregates inside cells. Regardless of chemical structure or physicochemical properties, bioaccumulation was consistently greater in macrophages than in epithelial cells. Microscopically, some self-assembled structures exhibited a pronounced, diattenuation anisotropy signal, evident by the differential absorption of linearly polarized light, at the peak absorbance wavelength of the phenazine core. The measured anisotropy was well above the background anisotropy of endogenous cellular components, reflecting the self-assembly of condensed, insoluble complexes of ordered phenazine molecules. Chemical variations introduced at the R-imino position of the phenazine core led to idiosyncratic effects on the compounds' bioaccumulation behavior, as well as on the morphology and organization of the resulting intracellular structures. Beyond clofazimine, these results demonstrate how the self-assembly of membrane-permeant, orally-bioavailable small molecule building blocks can endow cells with unnatural structural elements possessing chemical, physical and functional characteristics unlike those of other natural cellular components.
RESUMEN
Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of new modes of treating infection. We recently identified 1 (CCG-2979) as an inhibitor of the expression of streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with fibrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identified structural modifications that improve the solubility and metabolic stability of the scaffold. The efficacy of two new compounds 12c (CCG-203592) and 12k (CCG-205363) against biofilm formation in Staphylococcus aureus represents a promising additional mode of action for this novel class of compounds.