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1.
Eur Spine J ; 33(2): 438-443, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37934268

RESUMO

PURPOSE: Endoscopic endonasal approach (EEA) is the safest and most effective technique for odontoidectomy. Nevertheless, this kind of approach is yet not largely widespread. The aim of this study is to share with the scientific community some tips and tricks with our ten-year-old learned experience in endoscopic endonasal odontoidectomy (EEO), which remains a challenging surgical approach. MATERIAL AND METHODS: Our case series consists of twenty-one (10 males, 11 females; age range of 34-84 years) retrospectively analyzed patients with ventral spinal cord compression for non-reducible CVJ malformation, treated with EEA from July 2011 to March 2019. RESULTS: The results have recently been reported in a previous paper. The only intraoperative complication observed was intraoperative cerebrospinal fluid (CSF) leak (9.5%), without any sign of post-operative CSF leak. CONCLUSIONS: Considering our experience, EEO represents a valid and safe technique to decompress neural cervical structures. Despite its technical complexity, mainly due to the use of endoscope and the challenging surgical area, with this study we encourage the use of EEO displaying our experience-based surgical tips and tricks.


Assuntos
Descompressão Cirúrgica , Endoscopia , Feminino , Masculino , Humanos , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Criança , Estudos Retrospectivos , Vazamento de Líquido Cefalorraquidiano/etiologia , Vazamento de Líquido Cefalorraquidiano/cirurgia , Medula Espinal
2.
Eur Spine J ; 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38822150

RESUMO

PURPOSE: This retropective multicentric study aims to investigate the clinical applicability of the NSE score in the elderly, to verify the role of this tool as an easy help for decision making also for this class of patients. METHODS: All elderly patients (> 65 years) suffering from spinal metastases undergoing surgical or non-surgical treatment at the authors' Institutions between 2015 and 2022 were recruited. An agreement group (AG) and non-agreement group (NAG) were identified accordingly to the agreement between the NSE score indication and the performed treatment. Neurological status and axial pain were evaluated for both groups at follow-up (3 and 6 months). The same analysis was conducted specifically grouping patients older than 75 years. RESULTS: A strong association with improvement or preservation of clinical status (p < 0.001) at follow-up was obtained in AG. The association was not statistically significant in NAG at the 3-month follow-up (p 1.00 and 0.07 respectively) and at 6 months (p 0.293 and 0.09 respectively). The group of patients over 75 years old showed similar results in terms of statistical association between the agreement group and better outcomes. CONCLUSION: Far from the need or the aim to build dogmatic algorithms, the goal of preserving a proper performance status plays a key role in a modern oncological management: functional outcomes of the multicentric study group showed that the NSE score represents a reliable tool to establish the need for surgery also for elderly patients.

3.
J Enzyme Inhib Med Chem ; 38(1): 2245168, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37577806

RESUMO

The formal mechanism of linear mixed and non-competitive enzyme inhibition implies the binding of inhibitors to both the active site of the free enzyme in competition with the substrate, and to an allosteric site on the enzyme-substrate complex. However, it is evident from a review of the scientific literature that the two-site mechanism is frequently mistaken as the actual underlying mechanism of mixed inhibition. In this study, we conducted a comprehensive assessment of the mechanistic relevance of this type of inhibition using a statistical approach. By combining a statistical analysis of the inhibition cases documented in the BRENDA database with a theoretical investigation of inhibition models, we conclude that mixed inhibitors exclusively bind to the active site of enzymes. Hence ruling out any implication of allosteric sites and depriving the two-site model of any mechanistic relevance.


Assuntos
Inibidores Enzimáticos , Domínio Catalítico , Sítio Alostérico , Cinética , Sítios de Ligação , Inibidores Enzimáticos/química
4.
Int J Mol Sci ; 23(9)2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35563466

RESUMO

The Michaelis-Menten model of enzyme kinetic assumes the free ligand approximation, the steady-state approximation and the rapid equilibrium approximation. Analytical methods to model slow-binding inhibitors by the analysis of initial velocities have been developed but, due to their inherent complexity, they are seldom employed. In order to circumvent the complications that arise from the violation of the rapid equilibrium assumption, inhibition is commonly evaluated by pre-incubating the enzyme and the inhibitors so that, even for slow inhibitors, the binding equilibrium is established before the reaction is started. Here, we show that for long drug-target residence time inhibitors, the conventional analysis of initial velocities by the linear regression of double-reciprocal plots fails to provide a correct description of the inhibition mechanism. As a case study, the inhibition of acetylcholinesterase by galantamine, a drug approved for the symptomatic treatment of Alzheimer's disease, is reported. For over 50 years, analysis based on the conventional steady-state model has overlooked the time-dependent nature of galantamine inhibition, leading to an erroneous assessment of the drug potency and, hence, to discrepancies between biochemical data and the pharmacological evidence. Re-examination of acetylcholinesterase inhibition by pre-steady state analysis of the reaction progress curves showed that the potency of galantamine has indeed been underestimated by a factor of ~100.


Assuntos
Doença de Alzheimer , Inibidores da Colinesterase , Galantamina , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Galantamina/farmacologia , Galantamina/uso terapêutico , Humanos , Cinética
5.
J Enzyme Inhib Med Chem ; 33(1): 794-803, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29651884

RESUMO

Acetylcholinesterase inhibitors were introduced for the symptomatic treatment of Alzheimer's disease (AD). Among the currently approved inhibitors, donepezil (DNP) is one of the most preferred choices in AD therapy. The X-ray crystal structures of Torpedo californica AChE in complex with two novel rigid DNP-like analogs, compounds 1 and 2, have been determined. Kinetic studies indicated that compounds 1 and 2 show a mixed-type inhibition against TcAChE, with Ki values of 11.12 ± 2.88 and 29.86 ± 1.12 nM, respectively. The DNP rigidification results in a likely entropy-enthalpy compensation with solvation effects contributing primarily to AChE binding affinity. Molecular docking evidenced the molecular basis for the binding of compounds 1 and 2 to the active site of ß-secretase-1. Overall, these simplified DNP derivatives may represent new structural templates for the design of lead compounds for a more effective therapeutic strategy against AD by foreseeing a dual AChE and BACE-1 inhibitory activity.


Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Indanos/farmacologia , Piperidinas/farmacologia , Animais , Inibidores da Colinesterase/química , Cristalografia por Raios X , Donepezila , Relação Dose-Resposta a Droga , Indanos/química , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Piperidinas/química , Relação Estrutura-Atividade , Torpedo
6.
Comput Struct Biotechnol J ; 23: 34-42, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38089466

RESUMO

Activation-by-inhibition is a biochemical paradox seldom observed in exosite enzymes, wherein active site-bound inhibitors unexpectedly lead to enzyme activation. This intriguing phenomenon occurs at low, undersaturating substrate concentrations, posing a significant challenge in drug discovery, especially when targeting enzymes such as protein kinases, proteases, and other posttranslational modification enzymes. These enzymes often rely on accessory recognition sites known as exosites, which contribute to complex substrate binding mechanisms and unique kinetic behaviors. This study aims to provide a theoretical kinetic explanation for nonallosteric mechanism-based activation-by-inhibition, shedding light on the complexities of inhibiting exosite enzymes solely through active site targeting. Notably, the dual activator-inhibitor behavior of active site-bound inhibitors manifests in a nonmonotonic biphasic dose-response, emphasizing the importance of understanding the role of the inhibitor concentration at low substrate levels. Our findings underscore the potential widespread occurrence of activation by inhibition, a phenomenon that may have been overlooked in the past, and thus advocate for novel strategies in drug design that consider the impact of exosites on enzyme behavior to effectively target exosite enzymes.

7.
Protein Sci ; 33(5): e4977, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38591646

RESUMO

Chemical warfare nerve agents and pesticides, known as organophosphorus compounds inactivate cholinesterases (ChEs) by phosphorylating the serine hydroxyl group located at the active site of ChEs. Over the course of time, phosphorylation is followed by loss of an organophosphate-leaving group and the bond with ChEs becomes irreversible, a process known as aging. Differently, structurally related irreversible catalytic poisons bearing sulfur instead of phosphorus convert ChEs in its aged form only by covalently binding to the key catalytic serine. Kinetic and crystallographic studies of the interaction between Torpedo californica acetylcholinesterase (TcAChE) and a small organosulfonate, methanesulfonyl fluoride (MSF), indeed revealed irreversibly methylsulfonylated serine 200, to be isosteric with the bound aged sarin/soman analogues. The potent bulky reversible inhibitor 7-bis-tacrine (BTA) adopts, in the active site of the crystal structure of the MSF-enzyme adduct, a location and an orientation that closely resemble the one being found in the crystal structure of the BTA-enzyme complex. Remarkably, the presence of BTA accelerates the rate of methanesulfonylation by a factor of two. This unexpected result can be explained on the basis of two facts: i) the steric hindrance exerted by BTA to MSF in accessing the active site and ii) the acceleration of the MSF-enzyme adduct formation as a consequence of the lowering of the rotational and translational degrees of freedom in the proximity of the catalytic serine. It is well known that pralidoxime (2-Pyridine Aldoxime Methyl chloride, 2-PAM) alone or in the presence of the substrate acetylcholine cannot reactivate the active site serine of the TcAChE-MSF adduct. We show that the simultaneous presence of 2-PAM and the additional neutral oxime, 2-[(hydroxyimino)methyl]-l-methylimidazol (2-HAM), triggers the reactivation process of TcAChE within the hour timescale. Overall, our results pave the way toward the likely use of a cocktail of distinctive oximes as a promising recipe for an effective and fast reactivation of aged cholinesterases.


Assuntos
Acetilcolinesterase , Inibidores da Colinesterase , Compostos de Pralidoxima , Sulfonas , Taurina/análogos & derivados , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Acetilcolinesterase/química , Oximas/química , Serina
8.
World Neurosurg ; 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-39116943

RESUMO

PURPOSE: In surgical practice during endoscopic endonasal approach (EEA), Growth Hormone-secreting Pituitary Neuroendocrine Tumors (GH-secreting PitNET) patients show morphologic differences in the nasal cavities and sinuses, leading to a narrower surgical field and a carotid prominence and potentially increasing the complexity of the surgical and the risk of complications. The aim of the study is to evaluate the anatomical differences of the sphenoid sinus between patients with GH-secreting PitNETs and patients with non-functioning Pituitary Neuroendocrine Tumors (NF-PitNET) underwent EEA. METHODS: This is a monocentric retrospective study conducted at author's institution. The minimum intercarotid distance (ICS), the largest diameter of the sphenoid sinus (DSS) and the distance between vomer and clivus (VCD) were collected and compared. Presence, localization and course of intersphenoid sinus septum (ISS) were also evaluated. RESULTS: 100 consecutive patients were identified: 57 males (57%) and 43 females (43%), with a mean age of 55 years. 60 patients had NF-PitNET (60%) and 40 had GH-secreting PitNET (40%). GH-secreting PitNET group presented inferior values of ICD (16.8±3.94 mm vs. 20.4±3.94 mm, p<0.001), DSS (32.5±9.81 mm vs. 38.6±11.03 mm, p=0.006) and VCD (25.5±6.96 mm vs. 29.6±8.47 mm, p=0.012) compared to NF-PitNET group. ISS showed no differences between the two groups. CONCLUSION: ICD, DSS and VCD resulted smaller in acromegalic patients, confirming that patients with GH-secreting PitNETs have a narrower surgical field. A meticulous anatomical preoperative planning and neuronavigation are important to recognize the sphenoid anatomical landmarks in order to reduce the risk of complications, especially in acromegalic patients.

9.
Front Surg ; 10: 1158836, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37077862

RESUMO

Introduction: Evaluating the effects of indirect decompression obtained through lateral lumbar interbody fusion (LLIF) by clinical improvements and radiological parameters on MRI scans. Identifying predictors of better decompression and clinical outcome. Materials and methods: From 2016 to 2019, patients who underwent single- or double-level indirect decompression LLIF were consecutively reviewed. Radiological signs of indirect decompression were evaluated in preoperative and follow-up MRI studies and were subsequently correlated to clinical data, expressed as axial/radicular pain (VAS back/leg), index of disability (Oswestry Disability Index) and clinical severity of lumbar stenosis (Swiss Spinal Stenosis Questionnaire). Results: 72 patients were enrolled. The mean follow-up was 24 months. Differences in vertebral canal area (p < 0.001), height of the foramina (p < 0.001), thickness of the yellow ligament (p = 0.001) and anterior height of the interbody space (p = 0.02) were observed. Older age (p = 0.042), presence of spondylolisthesis (p = 0.042), presence of intra-articular facet effusion (p = 0.003) and posterior height of the implanted cage (p = 0.020) positively affected the increase of the canal area. Change in root canal area (p < 0.001), height of the implanted cage (p = 0.020) and younger age (p = 0.035) were predictive factors of root pain relief, while increased vertebral canal area (p = 0.020) and height of the interbody fusion cage (p = 0.023) positively affected the severity of clinical stenosis. Conclusions: LLIF indirect decompression showed both clinical and radiological improvements. Presence and degree of spondylolisthesis, presence of intra-articular facet effusion, age of the patient and height of the cage were predictive factors of major clinical improvements.

10.
Chem Biol Interact ; 365: 110092, 2022 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-35987277

RESUMO

Complex neurological disorders, including Alzheimer's disease, are one of the major therapeutic areas to which multitarget drug discovery strategies have been applied in the last twenty years. Due to the complex multifactorial etiopathogenesis of Alzheimer's disease, it has been proposed that to be successful the pharmaceutical agents should act on multiple targets in order to restore the complex disease network and to provide disease modifying effects. Here we report on the synthesis and the anticholinergic activity profiles of seven multitarget anti-Alzheimer compounds designed by combining galantamine, a well-known acetylcholinesterase inhibitor, with different peptide fragments endowed with inhibitory activity against BACE-1. A complementary approach based on molecular docking simulations of the galantamine-peptide derivatives in the active sites of acetylcholinesterase and of the related butyrylcholinesterase, as well as on inhibition kinetics, by global fitting of the reaction progress curves, allowed to gain insights into the enzyme-inhibitor mechanism of interaction. The resulting structure-activity relationships pave the way towards the design of more effective pharmacodynamic/pharmacokinetic multitarget inhibitors.


Assuntos
Doença de Alzheimer , Butirilcolinesterase , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Galantamina/farmacologia , Galantamina/uso terapêutico , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fragmentos de Peptídeos , Relação Estrutura-Atividade
11.
Cancers (Basel) ; 15(1)2022 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-36612085

RESUMO

Amino acid PET imaging has been used for a few years in the clinical and surgical management of gliomas with satisfactory results in diagnosis and grading for surgical and radiotherapy planning and to differentiate recurrences. Biological tumor volume (BTV) provides more meaningful information than standard MR imaging alone and often exceeds the boundary of the contrast-enhanced nodule seen in MRI. Since a gross total resection reflects the resection of the contrast-enhanced nodule and the majority of recurrences are at a tumor's margins, an integration of PET imaging during resection could increase PFS and OS. A systematic review of the literature searching for "PET" [All fields] AND "glioma" [All fields] AND "resection" [All fields] was performed in order to investigate the diffusion of integration of PET imaging in surgical practice. Integration in a neuronavigation system and intraoperative use of PET imaging in the primary diagnosis of adult high-grade gliomas were among the criteria for article selection. Only one study has satisfied the inclusion criteria, and a few more (13) have declared to use multimodal imaging techniques with the integration of PET imaging to intentionally perform a biopsy of the PET uptake area. Despite few pieces of evidence, targeting a biologically active area in addition to other tools, which can help intraoperatively the neurosurgeon to increase the amount of resected tumor, has the potential to provide incremental and complementary information in the management of brain gliomas. Since supramaximal resection based on the extent of MRI FLAIR hyperintensity resulted in an advantage in terms of PFS and OS, PET-based biological tumor volume, avoiding new neurological deficits, deserves further investigation.

12.
Brain Sci ; 11(11)2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34827434

RESUMO

BACKGROUND: Gliomas are among the most challenging pathologies for neurosurgeons due to their infiltrative and recurrent nature in functionally relevant regions. Current knowledge confirms that gross total resection highly influence survival in patient with glioma. However, surgery performed in eloquent brain area, could seriously compromise the quality of life in patient with reduced life expectancy even more if it concerns the language function. METHODS: 18 right-handed patients with perisylvian gliomas on the left hemisphere were prospectively analyzed over a period of 12 months. Standardized preoperative Diffusion-Tensor-Imaging based tractography of the five main language Tracts (Arcuate Fasciculus, Frontal Aslant Tract, Inferior Fronto-Occipital Fasciculus, Inferior Longitudinal Fasciculus, Uncinate Fasciculus) was navigated during the surgical procedure. Using a validated method, correlations were made between the pre-operative fascicles and their possible infiltration and surgical damage. The language status was assessed using the Aachen Aphasia Test. RESULTS: In all nine patients who developed a permanent disorder there was pre-operative involvement of at least one fascicle and resection of at least one of these. In this way, areas of high risk of permanent language damage have emerged as a result of surgical injury: the temporoparietal junction, the middle portion of the FAT and the temporal stem. CONCLUSIONS: Navigated tractography has proven to be a user-friendly tool that can assess perioperative risk, guide surgical resection, and help the neurosurgeon to find that balance between tumor resection and function preservation.

13.
Clin Neurol Neurosurg ; 197: 106162, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32890893

RESUMO

INTRODUCTION: Several hematological factors, such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), prognostic nutrition index (PNI) and albumin-to-globulin ratio (AGR), have been highlighted as systemic worse prognostic parameters for the outcome in gliomas. The aim of this study is to identify some pre-operative routinely blood tests as predictive parameters for the Overall Survival (OS) and Progression Free Survival (PFS) in glioblastoma (GBM). MATERIALS AND METHODS: From January 2013 to April 2019, 124 patients operated for glioblastoma were analyzed. Data were collected regarding age, sex, Karnofsky performance status (KPS), IDH status, the extent of resection (EOR) and adjuvant therapy. The hematological parameters were collected at admission: neutrophils, lymphocytes and platelets, hemoglobin, lactate dehydrogenase, albumin, NLR, PLR, AGR and PNI. The OS and the PFS were considered as the end-point for the evaluation of the predictive factors. RESULTS: A pre-operative neutrophil count > 7 × 109/L was a worse prognostic factor for OS and PFS at univariate analysis (p = 0.004 and p = 0.025), as well as hypo-albuminemia. Thrombocytosis, lymphopenia and NLR > 4 were associated to a worse OS, at uni- and multivariate analysis, resulting as poor predictive parameters, independently to EOR, the IDH mutation and the adjuvant therapy. CONCLUSIONS: Still nowadays there are no sensitive or specific hematological markers which are routinely applied for detecting and monitoring the treatment-response and the prognosis of glioblastoma. In our study, a pre-operative low cost and widely used blood markers, such as NLR, lymphocytes and platelets could be predictable prognostic factors for the Overall Survival of patients affected by glioblastomas.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/cirurgia , Glioblastoma/diagnóstico , Glioblastoma/cirurgia , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/epidemiologia , Feminino , Glioblastoma/sangue , Glioblastoma/epidemiologia , Testes Hematológicos , Humanos , Masculino , Pessoa de Meia-Idade , Cuidados Pré-Operatórios , Estudos Retrospectivos , Análise de Sobrevida , Resultado do Tratamento
14.
Eur J Med Chem ; 168: 491-514, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851693

RESUMO

A combination of tacrine and tryptophan led to the development of a new family of heterodimers as multi-target agents with potential to treat Alzheimer's disease. Based on the in vitro biological profile, compound S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), demonstrating balanced IC50 values of 6.3 and 9.1 nM, respectively. For all the tacrine-tryptophan heterodimers, favorable inhibitory effect on hAChE as well as on hBChE was coined to the optimal spacer length ranging from five to eight carbon atoms between these two pharmacophores. S-K1035 also showed good ability to inhibit Aß42 self-aggregation (58.6 ±â€¯5.1% at 50 µM) as well as hAChE-induced Aß40 aggregation (48.3 ±â€¯6.3% at 100 µM). The X-ray crystallographic analysis of TcAChE in complex with S-K1035 pinpointed the utility of the hybridization strategy applied and the structures determined with the two K1035 enantiomers in complex with hBChE could explain the higher inhibition potency of S-K1035. Other in vitro evaluations predicted the ability of S-K1035 to cross blood-brain barrier and to exert a moderate inhibition potency against neuronal nitric oxide synthase. Based on the initial promising biochemical data and a safer in vivo toxicity compared to tacrine, S-K1035 was administered to scopolamine-treated rats being able to dose-dependently revert amnesia.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/farmacologia , Fármacos Neuroprotetores/farmacologia , Tacrina/farmacologia , Triptofano/farmacologia , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Animais , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Humanos , Ligantes , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Agregados Proteicos/efeitos dos fármacos , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Tacrina/química , Triptofano/química
15.
Biochim Biophys Acta ; 1752(2): 197-201, 2005 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-16120484

RESUMO

We have recently purified an intracellular carboxylesterase encoded by the open reading frame PA3859 of Pseudomonas aeruginosa. Among proteins showing a significant sequence homology with PA3859 the in vivo function is only known for the human acyl-protein thioesterase I that is involved in the deacylation of Galpha proteins. The crystal structure determination of P. aeruginosa carboxylesterase is expected to provide insights into its physiological role. Therefore, the PA3859 gene was cloned and heterologously expressed in Escherichia coli as N-terminally 6xHis tagged recombinant protein. Here, we present the crystallization, X-ray diffraction analysis and phasing of this enzyme. Two crystal forms were obtained by the hanging drop vapor diffusion method. Crystals of form I belong to the space group P2(1) with cell dimensions of a=65.65, b=50.55, c=142.55 A, beta=92.9 degrees and diffracted, upon flash annealing, up to a resolution of 2.9 A. Two dimers are present in the asymmetric unit. Crystals of form II belong to space group P2(1)2(1)2, with unit cell dimensions of a=96.42, b=96.36, c=68.04 A and diffracted up to 2.1 A resolution. One dimer is present in the asymmetric unit.


Assuntos
Carboxilesterase/química , Pseudomonas aeruginosa/química , Cristalografia por Raios X , Difração de Raios X
16.
J Med Chem ; 59(1): 114-31, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26632651

RESUMO

Twenty-six new tacrine-benzofuran hybrids were designed, synthesized, and evaluated in vitro on key molecular targets for Alzheimer's disease. Most hybrids exhibited good inhibitory activities on cholinesterases and ß-amyloid self-aggregation. Selected compounds displayed significant inhibition of human ß-secretase-1 (hBACE-1). Among the 26 hybrids, 2e showed the most interesting profile as a subnanomolar selective inhibitor of human acetylcholinesterase (hAChE) (IC50 = 0.86 nM) and a good inhibitor of both ß-amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%, respectively) and hBACE-1 activity (IC50 = 1.35 µM). Kinetic studies showed that 2e acted as a slow, tight-binding, mixed-type inhibitor, while X-ray crystallographic studies highlighted the ability of 2e to induce large-scale structural changes in the active-site gorge of Torpedo californica AChE (TcAChE), with significant implications for structure-based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice. Finally, 2e administration did not exhibit significant hepatotoxicity.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Benzofuranos/química , Nootrópicos/química , Tacrina/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Comportamento Animal/efeitos dos fármacos , Benzofuranos/síntese química , Benzofuranos/farmacologia , Linhagem Celular , Sobrevivência Celular , Doença Hepática Induzida por Substâncias e Drogas/patologia , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Modelos Moleculares , Nootrópicos/síntese química , Nootrópicos/farmacologia , Relação Estrutura-Atividade , Tacrina/síntese química , Tacrina/farmacologia , Torpedo
17.
J Med Chem ; 57(20): 8576-89, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25259726

RESUMO

We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-ß (Aß) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Aß. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Antioxidantes/farmacologia , Inibidores da Colinesterase/farmacologia , Quinonas/química , Tacrina/química , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Antioxidantes/química , Barreira Hematoencefálica/efeitos dos fármacos , Técnicas de Química Sintética , Inibidores da Colinesterase/química , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Células Hep G2/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Terapia de Alvo Molecular , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar
18.
Chem Biol Interact ; 203(1): 129-34, 2013 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-23073172

RESUMO

We are evaluating a facilitative transport strategy to move oximes across the blood brain barrier (BBB) to reactivate inhibited brain acetylcholinesterase (AChE). We selected glucose (Glc) transporters (GLUT) for this purpose as these transporters are highly represented in the BBB. Glc conjugates have successfully moved drugs across the BBB and previous work has shown that Glc-oximes (sugar-oximes, SOxs) can reduce the organophosphonate induced hypothermia response. We previously evaluated the reactivation potential of Glc carbon C-1 SOxs. Here we report the reactivation parameters for VX- and GB-inhibited human (Hu) AChE of the best SOx (13c) and our findings that the kinetics are similar to those of the parent oxime. Although crystals of Torpedo californica AChE were produced, neither soaked or co-crystallized experiments were successful at concentrations below 20mM 13c, and higher concentrations cracked the crystals. 13c was non-toxic to neuroblastoma and kidney cell lines at 12-18 mM, allowing high concentrations to be used in a BBB kidney cell model. The transfer of 13c from the donor side was asymmetric with the greatest loss of 13c from the apical- or luminal-treated side. There was no apparent transfer from the basolateral side. The 13cP(app) results indicate a 'low' transport efficiency; however, mass accounting revealed only a 20% recovery from the apical dose in which high concentrations were found in the cell lysate fraction. Molecular modeling of 13c through the GLUT-1 channel demonstrated that transport of 13c was more restricted than Glc. Selected sites were compared and the 13c binding energies were greater than two times those of Glc.


Assuntos
Barreira Hematoencefálica , Reativadores da Colinesterase/farmacocinética , Oximas/farmacocinética , Acetilcolinesterase/metabolismo , Animais , Transporte Biológico Ativo , Reativadores da Colinesterase/química , Reativadores da Colinesterase/farmacologia , Reativadores da Colinesterase/toxicidade , Avaliação Pré-Clínica de Medicamentos , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/metabolismo , Humanos , Cinética , Modelos Biológicos , Modelos Moleculares , Oximas/química , Oximas/farmacologia , Oximas/toxicidade , Torpedo
19.
Am J Trop Med Hyg ; 87(1): 153-61, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22764307

RESUMO

Pyrazinamidase of Mycobacterium tuberculosis catalyzes the conversion of pyrazinamide to the active molecule pyrazinoic acid. Reduction of pyrazinamidase activity results in a level of pyrazinamide resistance. Previous studies have suggested that pyrazinamidase has a metal-binding site and that a divalent metal cofactor is required for activity. To determine the effect of divalent metals on the pyrazinamidase, the recombinant wild-type pyrazinamidase corresponding to the H37Rv pyrazinamide-susceptible reference strain was expressed in Escherichia coli with and without a carboxy terminal. His-tagged pyrazinamidase was inactivated by metal depletion and reactivated by titration with divalent metals. Although Co(2+), Mn(2+), and Zn(2+) restored pyrazinamidase activity, only Co(2+) enhanced the enzymatic activity to levels higher than the wild-type pyrazinamidase. Cu(2+), Fe(2+), Fe(3+), and Mg(2+) did not restore the activity under the conditions tested. Various recombinant mutated pyrazinamidases with appropriate folding but different enzymatic activities showed a differential pattern of recovered activity. X-ray fluorescence and atomic absorbance spectroscopy showed that recombinant wild-type pyrazinamidase expressed in E. coli most likely contained Zn. In conclusion, this study suggests that M. tuberculosis pyrazinamidase is a metalloenzyme that is able to coordinate several ions, but in vivo, it is more likely to coordinate Zn(2+). However, in vitro, the metal-depleted enzyme could be reactivated by several divalent metals with higher efficiency than Zn.


Assuntos
Amidoidrolases/metabolismo , Metais/farmacologia , Mycobacterium tuberculosis/enzimologia , Amidoidrolases/química , Dicroísmo Circular , Cinética , Modelos Moleculares , Espectrofotometria Atômica
20.
Biochimie ; 92(12): 1787-92, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20850500

RESUMO

The open reading frame PA3859 of Pseudomonas aeruginosa encodes an intracellular carboxylesterase belonging to a group of microbial enzymes (EC 3.1.1.1) that catalyze the hydrolysis of aliphatic and aromatic esters with a broad substrate specificity. With few exceptions, for this class of enzymes, belonging to the α/ß-hydrolase fold superfamily, very little information is available regarding their biochemical activity and in vivo function. The X-ray crystal structure of recombinant PA3859 has been determined for two crystal forms (space groups P2(1) and P2(1)2(1)2). The kinetic properties of the enzyme were studied using p-nitrophenyl esters as substrates and data fitted to a surface dilution mixed micelle kinetic model. Enzymatic assays and computational docking simulations, pinpointed the enzyme's preference for esters of palmitic and/or stearic acids and provided insights into the enzyme-substrate favorable binding modes.


Assuntos
Proteínas de Bactérias/metabolismo , Carboxilesterase/metabolismo , Ácidos Graxos/metabolismo , Pseudomonas aeruginosa/enzimologia , Algoritmos , Proteínas de Bactérias/química , Biocatálise , Carboxilesterase/química , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Ácidos Graxos/química , Hidrólise , Cinética , Modelos Moleculares , Nitrofenóis/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato
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