RESUMO
The MYC family of oncogenes (MYC, MYCN, and MYCL) encodes a basic helix-loop-helix leucine zipper (bHLHLZ) transcriptional regulator that is responsible for moving the cell through the restriction point. Through the HLHZIP domain, MYC heterodimerizes with the bHLHLZ protein MAX, which enables this MYC-MAX complex to bind to E-box regulatory DNA elements thereby controlling transcription of a large group of genes and their proteins. Translationally, MYC is one of the foremost oncogenic targets, and deregulation of expression of the MYC family gene/proteins occurs in over half of all human tumors and is recognized as a hallmark of cancer initiation and maintenance. Additionally, unexpected roles for this oncoprotein have been found in cancers that nominally have a non-MYC etiology. Although MYC is rarely mutated, its gain of function in cancer results from overexpression or from amplification. Moreover, MYC is a pleiotropic transcription factor possessing broad pathogenic prominence making it a coveted cancer target. A widely held notion within the biomedical research community is that the reliable modulation of MYC represents a tremendous therapeutic opportunity given its role in directly potentiating oncogenesis. However, the MYC-MAX heterodimer interaction contains a large surface area with a lack of well-defined binding sites creating the perception that targeting of MYC-MAX is forbidding. Here, we discuss the biochemistry behind MYC and MYC-MAX as it relates to cancer progression associated with these transcription factors. We also discuss the notion that MYC should no longer be regarded as undruggable, providing examples that a therapeutic window is achievable despite global MYC inhibition.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Neoplasias , Humanos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteínas Proto-Oncogênicas c-myc/química , Fatores de Transcrição/metabolismo , Oncogenes , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Cocaine is a highly addictive drug that has seen a steady uptrend causing severe health problems worldwide. Currently, there are no approved therapeutics for treating cocaine use disorder; hence, there is an urgent need to identify new medications. Immunopharmacotherapeutics is a promising approach utilizing endogenous antibodies generated through active vaccination, and if properly programmed, can blunt a drug's psychoactive and addictive effects. However, drug vaccine efficacy has largely been limited by the modest levels of antibodies induced. Herein, we explored an adjuvant system consisting of a polyphosphazene macromolecule, specifically poly[di(carboxylatoethylphenoxy)-phosphazene] (PCEP), a biocompatible synthetic polymer that was solicited for improved cocaine conjugate vaccine delivery performance. Our results demonstrated PCEP's superior assembling efficiency with a cocaine hapten as well as with the combined adjuvant CpG oligodeoxynucleotide (ODN). Importantly, this combination led to a higher titer response, balanced immunity, successful sequestering of cocaine in the blood, and a reduction in the drug in the brain. Moreover, a PCEP-cocaine conjugate vaccine was also found to function well via intranasal administration, where its efficacy was demonstrated through the antibody titer, affinity, mucosal IgA production, and a reduction in cocaine's locomotor activity. Overall, a comprehensive evaluation of PCEP integrated within a cocaine vaccine established an advance in the use of synthetic adjuvants in the drugs of abuse vaccine field.
Assuntos
Cocaína , Adjuvantes Imunológicos , Adjuvantes Farmacêuticos/farmacologia , Compostos Organofosforados , Polímeros , Desenvolvimento de Vacinas , Vacinas ConjugadasRESUMO
Unintentional overdose deaths related to opioids and psychostimulants have increased in prevalence due to the adulteration of these drugs with fentanyl. Synergistic effects between illicit compounds and fentanyl cause aggravated respiratory depression, leading to inadvertent fatalities. Traditional small-molecule therapies implemented in the expanding opioid epidemic present numerous problems since they interact with the same opioid receptors in the brain as the abused drugs. In this study, we report an optimized dual hapten for use as an immunopharmacotherapeutic tool in order to develop antibodies capable of binding to fentanyl-contaminated heroin in the periphery, thus impeding the drugs' psychoactive effects on the central nervous system. This vaccine produced antibodies with nanomolar affinities and effectively blocked opioid analgesic effects elicited by adulterated heroin. These findings provide further insight into the development of chemically contiguous haptens for broad-spectrum immunopharmacotherapies against opioid use disorders.
Assuntos
Overdose de Drogas/prevenção & controle , Fentanila/imunologia , Haptenos/imunologia , Heroína/efeitos adversos , Heroína/química , Vacinas/imunologia , Animais , Contaminação de Medicamentos , Overdose de Drogas/mortalidade , Fentanila/efeitos adversos , Fentanila/química , Humanos , Camundongos , Transtornos Relacionados ao Uso de OpioidesRESUMO
We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
Assuntos
Desenvolvimento de Medicamentos , Corantes Fluorescentes/farmacologia , Ensaios de Triagem em Larga Escala , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Relação Estrutura-AtividadeRESUMO
The United States is in the midst of an unprecedented epidemic of opioid substance use disorder, and while pharmacotherapies including opioid agonists and antagonists have shown success, they can be inadequate and frequently result in high recidivism. With these challenges facing opioid use disorder treatments immunopharmacotherapy is being explored as an alternative therapy option and is based upon antibody-opioid sequestering to block brain entry. Development of a heroin vaccine has become a major research focal point; however, producing an efficient vaccine against heroin has been particularly challenging because of the need to generate not only a potent immune response but one against heroin and its multiple psychoactive molecules. In this study, we explored the consequence of regioselective deuteration of a heroin hapten and its impact upon the immune response against heroin and its psychoactive metabolites. Deuterium (HdAc) and cognate protium heroin (HAc) haptens were compared head to head in an inclusive vaccine study. Strikingly the HdAc vaccine granted greater efficacy in blunting heroin analgesia in murine behavioral models compared to the HAc vaccine. Binding studies confirmed that the HdAc vaccine elicited both greater quantities and equivalent or higher affinity antibodies toward heroin and 6-AM. Blood-brain biodistribution experiments corroborated these affinity tests. These findings suggest that regioselective hapten deuteration could be useful for the resurrection of previous drug of abuse vaccines that have met limited success in the past.
Assuntos
Haptenos/química , Haptenos/imunologia , Heroína/imunologia , Vacinas/química , Vacinas/imunologia , Deutério/química , Heroína/química , Conformação MolecularRESUMO
Botulinum neurotoxin serotype A (BoNT/A), marketed commercially as Botox, is the most toxic substance known to man with an estimated intravenous lethal dose (LD50) of 1-2 ng/kg in humans. Despite its widespread use in cosmetic and medicinal applications, no postexposure therapeutics are available for the reversal of intoxication in the event of medical malpractice or bioterrorism. Accordingly, the Centers for Disease Control and Prevention categorizes BoNT/A as a Category A pathogen, posing the highest risk to national security and public health as a result of the ease with which BoNT/A can be weaponized and disseminated. BoNT/A-mediated lethality results from neurons impeded from releasing acetylcholine, which ultimately causes muscle paralysis and possible death by asphyxiation with the loss of diaphragm function. Currently, the only available respite for BoNT/A poisoning is antibody-based therapy; however, this intervention is only effective within 12-24 h postexposure. Small molecule therapeutics remain the only opportunity to reverse BoNT/A intoxication after neuronal poisoning and are urgently needed. Nevertheless, no small molecule BoNT/A inhibitors have reached the clinic or even advanced to clinical trials. This Account highlights the accomplishments and existing challenges facing BoNT/A drug discovery today. Using the comprehensive body of work from our laboratory, we illustrate our nearly two-decade endeavor to discover a clinically relevant BoNT/A inhibitor. Specifically, a discussion on the identification and characterization of new chemical leads, the development of in vitro and in vivo assays, and pertinent discoveries in BoNT/A structural biology related to small molecule inhibition is presented. Lead discovery efforts in our laboratory have leveraged both in vitro high-throughput screening and rational design, and an array of mechanistic strategies for inhibiting BoNT/A has been discovered, including noncovalent inhibition, metal-binding active site inhibition, covalent inhibition, and α- and ß-exosite inhibition. We contrast the strengths and weaknesses of each of these mechanistic strategies and propose the most favorable approach for success. Finally, we discuss multiple serendipitous discoveries of antibotulism small molecules with alternative mechanisms of action. Remaining challenges facing clinically relevant BoNT/A inhibition are presented and analyzed, including the current inability to reconcile toxin half-life (months to greater than one year) in neurons with in vivo pharmaceutical lifetimes and reoccurring inconsistencies between in vitro, cellular, and in vivo translation. Our Account of BoNT/A chemical research emphasizes the present accomplishments and critically analyzes the remaining obstacles for drug discovery. Importantly, we call for an increased focus on the discovery of safe and effective covalent inhibitors of BoNT/A that compete with the inherent half-life of the toxin.
Assuntos
Toxinas Botulínicas Tipo A/antagonistas & inibidores , Botulismo/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Animais , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/metabolismo , Domínio Catalítico , Humanos , Camundongos , Ligação Proteica , Proteólise , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Heroin overdose and addiction remain significant health and economic burdens in the world today costing billions of dollars annually. Moreover, only limited pharmacotherapeutic options are available for treatment of heroin addiction. In our efforts to combat the public health threat posed by heroin addiction, we have developed vaccines against heroin. To expand upon our existing heroin-vaccine arsenal, we synthesized new aryl and alkyl sulfonate ester haptens; namely aryl-mono-sulfonate (HMsAc) and Aryl/alkyl-di-sulfonate (H(Ds)2) as carboxyl-isosteres of heroin then compared them to our model heroin-hapten (HAc) through vaccination studies. Heroin haptens were conjugated to the carrier protein CRM197 and the resulting CRM-immunoconjugates were used to vaccinate Swiss Webster mice following an established immunization protocol. Binding studies revealed that the highest affinity anti-heroin antibodies were generated by the HMsAc vaccine followed by the HAc and H(Ds)2 vaccines, respectively (HMsAc > HAcâ«HDs2). However, neither the HMsAc nor H(Ds)2 vaccines were able to generate high affinity antibodies to the psychoactive metabolite 6-acetyl morphine (6-AM), in comparison to the HAc vaccine. Blood brain bio-distribution studies supported these binding results with vaccine efficiency following the trend HAc > HMsAc â« H(Ds)2 The work described herein provides insight into the use of hapten-isosteric replacement in vaccine drug design.
Assuntos
Alcanossulfonatos/química , Desenho de Fármacos , Haptenos/química , Heroína/química , Vacinas Sintéticas/imunologia , Animais , Anticorpos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Encéfalo/metabolismo , Haptenos/imunologia , Heroína/imunologia , Camundongos , Vacinas Sintéticas/sangue , Vacinas Sintéticas/metabolismoRESUMO
The present United States opioid crisis requires urgent and innovative scientific intervention. This perspective highlights a role for the chemical sciences by expounding upon three key research areas identified as priorities by the National Institute on Drug Abuse (NIDA). Specifically, important advances in chemical interventions for overdose reversal, strategies for opioid use disorder (OUD) treatment, including immunopharmacotherapies, and next-generation alternatives for pain management will be discussed. Ultimately, progress made will be presented in light of remaining challenges for the field.
Assuntos
Analgésicos Opioides/efeitos adversos , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Analgésicos Opioides/química , Overdose de Drogas , Humanos , National Institute on Drug Abuse (U.S.) , Estados UnidosRESUMO
Botulinum neurotoxin serotype A (BoNT/A) causes a debilitating and potentially fatal illness known as botulism. The toxin is also a bioterrorism threat, yet no pharmacological antagonist to counteract its effects has reached clinical approval. Existing strategies to negate BoNT/A intoxication have looked to antibodies, peptides, or organic small molecules as potential therapeutics. In this work, a departure from the traditional drug discovery mindset was pursued, in which the enzyme's susceptibility to metal ions was exploited. A screen of a series of metal salts showed marked inhibitory activity of group 11 and 12 metals against the BoNT/A light chain (LC) protease. Enzyme kinetics revealed that copper (I) and (II) cations displayed noncompetitive inhibition of the LC (Ki ≈ 1 µM), while mercury (II) cations were 10-fold more potent. Crystallographic and mutagenesis studies elucidated a key binding interaction between Cys165 on BoNT/A LC and the inhibitory metals. As potential copper prodrugs, ligand-copper complexes were examined in a cell-based model and were found to prevent BoNT/A cleavage of the endogenous protein substrate, SNAP-25, even at low µM concentrations of complexes. Further investigation of the complexes suggested a bioreductive mechanism causing intracellular release of copper, which directly inhibited the BoNT/A protease. In vivo experiments demonstrated that copper (II) dithiocarbamate and bis(thiosemicarbazone) complexes could delay BoNT/A-mediated lethality in a rodent model, indicating their potential for treating the harmful effects of BoNT/A intoxication. Our studies illustrate that metals can be therapeutically viable enzyme inhibitors; moreover, enzymes that share homology with BoNT LCs may be similarly targeted with metals.
Assuntos
Toxinas Botulínicas Tipo A/antagonistas & inibidores , Complexos de Coordenação/farmacologia , Cobre/farmacologia , Inibidores de Proteases/farmacologia , Tiocarbamatos/farmacologia , Tiossemicarbazonas/farmacologia , Animais , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/toxicidade , Complexos de Coordenação/química , Cobre/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Cinética , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Inibidores de Proteases/química , Relação Estrutura-Atividade , Tiocarbamatos/química , Tiossemicarbazonas/químicaRESUMO
The Neglected Tropical Disease onchocerciasis is a parasitic disease. Despite many control programmes by the World Health Organization (WHO), large communities in West and Central Africa are still affected. Besides logistic challenges during biannual mass drug administration, the lack of a robust, point-of-care diagnostic is limiting successful eradication of onchocerciasis. Towards the implementation of a non-invasive and point-of-care diagnostic, we have recently reported the discovery of the biomarker N-acetyltyramine-O-glucuronide (NATOG) in human urine samples using a metabolomics-mining approach. NATOG's biomarker value was enhanced during an investigation in a rodent model. Herein, we further detail the specificity of NATOG in active onchocerciasis infections as well as the co-infecting parasites Loa loa and Mansonella perstans. Our results measured by liquid chromatography coupled with mass spectrometry (LC-MS) reveal elevated NATOG values in mono- and co-infection samples only in the presence of the nematode Onchocerca volvulus. Metabolic pathway investigation of l-tyrosine/tyramine in all investigated nematodes uncovered an important link between the endosymbiotic bacterium Wolbachia and O. volvulus for the biosynthesis of NATOG. Based on these extended studies, we suggest NATOG as a biomarker for tracking active onchocerciasis infections and provide a threshold concentration value of NATOG for future diagnostic tool development.
Assuntos
Glucuronídeos/urina , Espectrometria de Massas/métodos , Doenças Negligenciadas/urina , Onchocerca volvulus/isolamento & purificação , Oncocercose/urina , Tiramina/análogos & derivados , Animais , Biomarcadores/urina , Cromatografia Líquida/métodos , Glucuronídeos/metabolismo , Humanos , Limite de Detecção , Metabolômica/métodos , Doenças Negligenciadas/metabolismo , Onchocerca volvulus/metabolismo , Oncocercose/metabolismo , Tiramina/metabolismo , Tiramina/urinaRESUMO
Carbohydrate antigens displaying Galα(1,3)Gal epitopes are recognized by naturally occurring antibodies in humans. These anti-Gal antibodies comprise up to 1% of serum IgG and have been viewed as detrimental as they are responsible for hyperacute organ rejections. In order to model this condition, α(1,3)galactosyltransferase-knockout mice are inoculated against the Galα(1,3)Gal epitope. In our study, two α-Gal trisaccharide epitopes composed of either Galα(1,3)Galß(1,4)GlcNAc or Galα(1,3)Galß(1,4)Glc linked to a squaric acid ester moiety were examined for their ability to elicit immune responses in KO mice. Both target epitopes were synthesized using a two-component enzymatic system using modified disaccharide substrates containing a linker moiety for coupling. While both glycoconjugate vaccines induced the required high anti-Gal IgG antibody titers, it was found that this response had exquisite specificity for the Galα(1,3)Galß(1,4)GlcNAc hapten used, with little cross reactivity with the Galα(1,3)Galß(1,4)Glc hapten. Our findings indicate that while homogenous glycoconjugate vaccines provide high IgG titers, the carrier and adjuvanting factors can deviate the specificity to an antigenic determinant outside the purview of interest.
Assuntos
Desenho de Fármacos , Epitopos/química , Epitopos/imunologia , Trissacarídeos/imunologia , Técnicas de Química Sintética , Haptenos/imunologiaRESUMO
Although cocaine abuse and addiction continue to cause serious health and societal problems, an FDA-approved medication to treat cocaine addiction has yet to be developed. Employing a pharmacokinetic strategy, an anticocaine vaccine provides an attractive avenue to address these issues; however, current vaccines have shown varying degrees of efficacy, indicating that further formulation is necessary. As a means to improve vaccine efficacy, we examined the effects of varying anticocaine vaccine formulations by combining a Toll-like receptor 9 (TLR9) agonist with a TLR5 agonist in the presence of alum. The TLR9 agonist used was cytosine-guanine oligodeoxynucleotide 1826 (CpG 1826), while the TLR5 agonist was flagellin (FliC). Formulations with the TLR9 agonist elicited superior anticocaine antibody titers and blockade of hyperlocomotor effects compared to vaccines without CpG 1826. This improvement was seen regardless of whether the TLR5 agonist, FliC, or the nonadjuvanting Tetanus Toxoid (TT) was used as the carrier protein. Additional insights into the value of FliC as a carrier versus adjuvant was also investigated by generating two unique formats of the protein, wild-type and mutated flagellin (mFliC). While the mFliC conjugate retained its ability to stimulate mTLR5, it yielded reduced cocaine sequestration and functional blockade relative to FliC and TT. Overall, this work indicates that activation of TLR9 can improve the function of cocaine vaccines in the presence of TLR5 activation by FliC, with any potential additive effects limited by the inefficiency of FliC as a carrier protein as compared to TT.
Assuntos
Cocaína/imunologia , Animais , Cocaína/metabolismo , Ensaio de Imunoadsorção Enzimática , Flagelina/metabolismo , Masculino , Espectrometria de Massas , Radioimunoensaio , Receptor 5 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/metabolismo , Vacinas/imunologia , Vacinas/metabolismoRESUMO
There is currently no clinically-approved antidote for cocaine overdose. Efforts to develop a therapy via passive immunization have resulted in a human monoclonal antibody, GNCgzk, with a high affinity for cocaine (Kd=0.18nM). Efforts to improve the production of antibody manifolds based on this antibody are disclosed. The engineering of an HRV 3C protease cleavage site into the GNCgzk IgG has allowed for increased production of a F(ab')2 with a 20% superior capacity to reduce mortality for cocaine overdose in mice.
Assuntos
Anticorpos Monoclonais/imunologia , Cocaína/imunologia , Overdose de Drogas/terapia , Doença Aguda , Animais , Meia-Vida , Masculino , CamundongosRESUMO
Cocaine abuse is problematic, directly and indirectly impacting the lives of millions, and yet existing therapies are inadequate and usually ineffective. A cocaine vaccine would be a promising alternative therapeutic option, but efficacy is hampered by variable production of anticocaine antibodies. Thus, new tactics and strategies for boosting cocaine vaccine immunogenicity must be explored. Flagellin is a bacterial protein that stimulates the innate immune response via binding to extracellular Toll-like receptor 5 (TLR5) and also via interaction with intracellular NOD-like receptor C4 (NLRC4), leading to production of pro-inflammatory cytokines. Reasoning that flagellin could serve as both carrier and adjuvant, we modified recombinant flagellin protein to display a cocaine hapten termed GNE. The resulting conjugates exhibited dose-dependent stimulation of anti-GNE antibody production. Moreover, when adjuvanted with alum, but not with liposomal MPLA, GNE-FliC was found to be better than our benchmark GNE-KLH. This work represents a new avenue for exploration in the use of hapten-flagellin conjugates to elicit antihapten immune responses.
Assuntos
Cocaína/imunologia , Flagelina/química , Haptenos/química , Vacinas/imunologia , Adjuvantes Imunológicos/química , Compostos de Alúmen/química , Animais , Peso Corporal , Ensaio de Imunoadsorção Enzimática , Flagelina/imunologia , Haptenos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RadioimunoensaioRESUMO
The neglected tropical disease onchocerciasis affects more than 35 million people worldwide with over 95% in Africa. Disease infection initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious L3 larvae. New treatments and diagnostics are required to eradicate this parasitic disease. Herein, we describe that a previously discovered biomarker for onchocerciasis, N-acetyltyramine-O-glucuronide (NATOG) is also present in urine samples of jirds infected with the onchocerciasis model nematode Litomosoides sigmodontis. Increased NATOG values paralleled a progressing infection and demonstrated that quantification of NATOG in this rodent model can be utilized to track its infectivity. Moreover, our findings suggest how NATOG monitoring may be used for evaluating potential drug candidates.
Assuntos
Filarioidea/isolamento & purificação , Glucuronídeos/urina , Metaboloma , Oncocercose/patologia , Animais , Biomarcadores/urina , Filarioidea/crescimento & desenvolvimento , Filarioidea/fisiologia , Gerbillinae , Estágios do Ciclo de Vida , Oncocercose/parasitologia , Oncocercose/veterinária , Análise de Componente PrincipalRESUMO
Cocaine abuse remains prevalent worldwide and continues to be a major health concern; nonetheless, there is no effective therapy. Immunopharmacotherapy has emerged as a promising treatment strategy by which anti-cocaine antibodies bind to the drug blunting its effects. Previous passive immunization studies using our human monoclonal antibody, GNCgzk, resulted in protection against cocaine overdose and acute toxicity. To further realize the clinical potential of this antibody, a recombinant IgG form of the antibody has been produced in mammalian cells. This antibody displayed a high binding affinity for cocaine (low nanomolar) in line with the superior attributes of the GNCgzk antibody and reduced cocaine-induced ataxia in a cocaine overdose model.
Assuntos
Anticorpos Monoclonais/imunologia , Ataxia/tratamento farmacológico , Cocaína/imunologia , Animais , Ataxia/induzido quimicamente , Cocaína/antagonistas & inibidores , Modelos Animais de Doenças , Humanos , Imunoterapia , Camundongos , Conformação Molecular , Proteínas Recombinantes/imunologiaRESUMO
Xylazine has emerged as a primary adulterant in fentanyl, exacerbating the complexity of the opioid crisis. Yet, there is no approved drug that can reverse xylazine's pathophysiology. As a prelude to monoclonal antibodies being assessed as a viable therapeutic, a vaccine inquiry was conducted evaluating the immune response in reversing xylazine induced behavior effects.
Assuntos
Haptenos , Xilazina , Xilazina/química , Xilazina/farmacologia , Haptenos/química , Haptenos/imunologia , Animais , Vacinas Conjugadas/química , Vacinas Conjugadas/imunologia , CamundongosRESUMO
Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness.
Assuntos
Anti-Helmínticos/farmacologia , Quitina/antagonistas & inibidores , Quitinases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Onchocerca/efeitos dos fármacos , Oncocercose/tratamento farmacológico , Salicilanilidas/farmacologia , Animais , Anti-Helmínticos/química , Anti-Helmínticos/isolamento & purificação , Quitina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Humanos , Concentração Inibidora 50 , Muda/efeitos dos fármacos , Onchocerca/enzimologia , Onchocerca/crescimento & desenvolvimento , Salicilanilidas/química , Salicilanilidas/isolamento & purificação , Bibliotecas de Moléculas PequenasRESUMO
Carfentanil, the most potent of the fentanyl analogues, is at the forefront of synthetic opioid-related deaths, second to fentanyl. Moreover, the administration of the opioid receptor antagonist naloxone has proven inadequate for an increasing number of opioid-related conditions, often requiring higher/additional doses to be effective, as such interest in alternative strategies to combat more potent synthetic opioids has intensified. Increasing drug metabolism would be one strategy to detoxify carfentanil; however, carfentanil's major metabolic pathways involve N-dealkylation or monohydroxylation, which do not lend themselves readily to exogenous enzyme addition. Herein, we report, to our knowledge, the first demonstration that carfentanil's methyl ester when hydrolyzed to its acid was found to be 40,000 times less potent than carfentanil in activating the µ-opioid receptor. Physiological consequences of carfentanil and its acid were also examined through plethysmography, and carfentanil's acid was found to be incapable of inducing respiratory depression. Based upon this information, a hapten was chemically synthesized and immunized, allowing the generation of antibodies that were screened for carfentanil ester hydrolysis. From the screening campaign, three antibodies were found to accelerate the hydrolysis of carfentanil's methyl ester. From this series of catalytic antibodies, the most active underwent extensive kinetic analysis, allowing us to postulate its mechanism of hydrolysis against this synthetic opioid. In the context of potential clinical applications, the antibody, when passively administered, was able to reduce respiratory depression induced by carfentanil. The data presented supports further development of antibody catalysis as a biologic strategy to complement carfentanil overdose reversal.
RESUMO
Opioid use disorders and overdose have become a major public health concern in recent years. U-47700, a New psychoactive substances (NPS) opioid, also known as "pinky" or "pink" has been identified as a new threat in the drug supply because of its potency and abuse potential. Conjugate vaccines that can produce antibodies against target drug molecules have emerged as a promising tool to treat substance use disorders. Herein, we report the design, synthesis, and in vivo characterization of a U-47700 vaccine. The vaccine demonstrated favorable results with rodents producing elevated levels of antibody titer and sub-micromolar affinity to U-47700. In addition, antibodies generated by the vaccine effectively mitigated drug-induced effects by preventing the drug from penetrating the blood-brain barrier, which was verified by antinociception and drug biodistribution studies. The development of a vaccine against U-47700 and other NPS opioids contributes to the continued advancement of non-conventional pharmacological treatments to address the global opioid epidemic.