真菌多药耐药外排机制的研究进展

真菌多药耐药性是指真菌细胞对结构不同、作用靶点不同的药物同时具有耐药性的现象,是导致临床抗真菌治疗失败的重要原因之一。本文综述了酿酒酵母、条件致病真菌白假丝酵母、光滑假丝酵母和烟曲霉中多药耐药相关转运蛋白、药物外排机制以及基因表达调控网络的研究进展,旨在为深入了解真菌多药耐药性的机制、探讨克服多药耐药性的策略和提高抗真菌药物的药效提供参考。     

药物耐药性外排泵抑制剂的研究进展

外排泵抑制剂可以抑制耐药细菌和肿瘤细胞对进入胞体内药物的外排作用,提高对药物的敏感性.它在改善药物的药动学特性等方面也有广泛的作用.研制开发外排泵抑制剂,逆转现有药物的耐药性将是改善药物疗效的一种有效的治疗方法.简要阐述近年来外排泵抑制剂的研究进展.     

Inhibition of bacterial efflux pumps: a new strategy to combat increasing antimicrobial agent resistance

Resistance to multiple drugs in medically important bacteria results in therapeutic challenges for the clinician. The mechanisms by which bacteria evade the effects of antimicrobial agents are many, but in recent years it has become apparent that efflux is a significant means of resistance and probably explains the intrinsic resistance to numerous drugs observed in species such as Pseudomonas aeruginosa. Drug efflux is mediated by membrane-based hydrophobic proteins belonging to several distinct families, the members of which are related by structural characteristics, mechanism of action and energy source for the transport process. The multi-drug efflux transporters are particularly problematic as they are capable of extruding numerous structurally dissimilar drugs. Inhibition of these pumps, and even those with more limited substrate specificity, has been shown to decrease intrinsic resistance, reverse acquired resistance and reduce the emergence of mutants with higher-level target-based mutational resistance. Combining broad spectrum efflux pump inhibitors with current drugs that are pump substrates can recover clinically relevant activity of those compounds and thus may reduce the need for the discovery and development of new antimicrobial agents that are not pump substrates. Additional effort toward the identification, characterisation and determination of the clinical utility of efflux pump inhibitors is warranted.     

外排型转运体与肿瘤多药耐药

肿瘤多药耐药（MDR）是导致肿瘤化疗失败的主要原因之一。肿瘤MDR的机制有多种,其中外排型转运体的过表达是导致MDR的主要机制,因此研究外排型转运体介导的肿瘤MDR机制和发现可以逆转肿瘤MDR的抑制剂成为国内外研究的热点。就目前研究的3种三磷酸腺苷结合盒转运体：P-糖蛋白、多药耐药相关蛋白、乳腺癌耐药蛋白介导的MDR及逆转MDR的机制进行综述,以期为提高肿瘤治疗疗效提供依据。     

细菌外排机制及其抑制剂研究进展

细菌耐药性,尤其是多重耐药问题日益严重,给临床治疗带来极大的困难.而细菌外排机制是细菌产生多重耐药的重要机制之一.本文对细菌外排泵系统的组成、种类和几种常见耐药病原菌(包括大肠埃希菌、铜绿假单胞菌和金葡菌)中的主要外排泵,以及外排泵抑制剂的研究进展作简要综述.     

DbMDR: a relational database for multidrug resistance genes as potential drug targets

DbMDR is non-redundant reference database of multidrug resistance (MDR) genes and their orthologs acting as potential drug targets. Drug resistance is a common phenomenon of pathogens, creating a serious problem of inactivation of drugs and antibiotics resulting in occurrence of diseases. Apart from other factors, the MDR genes present in pathogens are shown to be responsible for multidrug resistance. Much of the unorganized information on MDR genes is scattered across the literature and other web resources. Thus, consolidation of such knowledge about MDR genes into one database will make the drug discovery research more efficient. Mining of text for MDR genes has resulted into a large number of publications but in scattered and unorganized form. This information was compiled into a database, which enables a user not only to look at a particular MDR gene but also to find out putative homologs based on sequence similarity, conserved domains, and motifs in proteins encoded by MDR genes more efficiently. At present, DbMDR database contains 2843 MDR genes characterized experimentally as well as functionally annotated with cross-referencing search support. The DbMDR database (http://203.190.147.116/dbmdr/) is a comprehensive resource for comparative study focused on MDR genes and metabolic pathway efflux pumps and intended to provide a platform for researchers for further research in drug resistance.     

微生物药物外排泵及其抑制剂研究

细菌自身的不断进化及抗生素的不合理使用导致了耐药性细菌的出现和蔓延，微生物的主动外排是导致细菌产生多重耐药的重要原因。微生物外排泵的研究是目前的热点之一，也被认为是开发新抗生素的理想靶标。本文从基因组和比较基因组水平对外排泵的类型、作用机制、表达调控及研究方法等方面概述，并结合正在开展的分枝杆菌的药物外排泵进行了比较基因组分析。这些工作有利于开发新的抗生素或抗生素增效剂。     

Kinetic Considerations for the Quantitative Assessment of Efflux Activity and Inhibition: Implications for Understanding and Predicting the Effects of Efflux Inhibition

Purpose Unexpected and complex experimental observations related to efflux transport have been reported in the literature. This work was conducted to develop relationships for efflux activity (PS_efflux) as a function of commonly studied kinetic parameters [permeability-surface area product (PS), efflux ratio (ER), degree of efflux inhibition (ϕ_i), 50% inhibitory concentration (IC₅₀), and Michaelis–Menten constant (K_m)]. Methods A three-compartment model (apical, cellular, and basolateral) was used to derive flux equations relating the initial rate of flux and steady-state mass transfer in the presence or absence of active efflux. Various definitions of efflux ratio (ER) were examined in terms of permeability-surface area products. The efflux activity (PS_efflux) was expressed in terms of ER and PS. The relationships between PS_efflux and PS, ER, ϕ_i, IC₅₀, and K_m were solved mathematically. Simulations and examples from the literature were used to illustrate the resulting mathematical relationships. Results The relationships derived according to a three-compartment model differed fundamentally from commonly accepted approaches for determining PS_efflux, ϕ_i, IC₅₀ and K_m. Based on the model assumptions and mathematical derivations, currently used mathematical relationships erroneously imply that efflux activity is proportional to change in PS (i.e., flux or P_app) and thus underestimate PS_efflux and ϕ_i, and overestimate IC₅₀ and K_m. Conclusions An understanding of the relationship between efflux inhibition and kinetic parameters is critical for appropriate data interpretation, standardization in calculating and expressing the influence of efflux transport, and predicting the clinical significance of efflux inhibition.     

Multidrug efflux pumps and their role in antibiotic and antiseptic resistance: a pharmacodynamic perspective

Introduction: Worrying levels of bacterial resistance have been reported worldwide involving the failure of many available antibiotic treatments. Multidrug resistance (MDR) in Gram-negative bacteria is often ascribed to the presence of multiple and different resistance mechanisms in the same strain. RND efflux pumps play a major role and are an attractive target to discover new antibacterial drugs.

Areas covered: This review discusses the prevalence of efflux pumps, their overexpression in clinical scenarios, their polyselectivity, their effect on the intracellular concentrations of various antibiotics associated with the alteration of the membrane permeability and their involvement in pathogenicity are discussed.

Expert opinion: Efflux pumps are new targets for the development of adjuvant in antibiotic treatments by of efflux pump inhibition. They may allow us to rejuvenate old antibiotics acting on their concentration inside the bacteria and thus potentiating their activity while blocking the release of virulence factors. It is a pharmacodynamic challenge to finalize new combined therapy.     

多药耐药相关蛋白及其在肿瘤耐药中的作用

多药耐药(multidrug resistance,MDR)是导致肿瘤患者化疗失败的主要原因。介导多药耐药的重要机制之一是多药耐药相关蛋白(multidrug resistance-associated proteins,MRPs)的表达增加。MRPs是一类ATP能量依赖型跨膜转运蛋白,是具有选择性和特异性的药物外排泵。本文主要针对MRPs的生理特征、结构特点、耐药谱特征及其逆转进行综述。     

Overcoming multiple drug resistance in cancer using polymeric micelles

ABSTRACT

Introduction: A major concern that limits the success of cancer chemotherapy is multidrug resistance (MDR). The drug resistance mechanisms are either host related or tumor related. The host tumor interacting factors also contribute to MDR. Multifunctional polymeric micelles offer several advantages in circumventing MDR due to their design, selectivity, and stability in cancer microenvironment.

Areas covered: The review is broadly divided into two parts: the first part covers MDR and its mechanisms; the second part covers multifunctional polymeric micelles in combating MDR through its state-of-the-art design. This part covers various strategies like use of P-gp transporter inhibitors, TPGS, pH & thermo-sensitive, and siRNA for selectivity of PMs against multidrug-resistant tumors.

Expert opinion: Numerous approaches have been tested using polymeric micelles to overcome MDR tumors. However, these are either limited to only in-vitro investigations and/or preliminary preclinical models and do not investigate the underlying biological mechanism. Hence, there exists an unmet need to perform fundamental research that focuses on studying the underlying mechanism and preclinical/clinical testing of the micellar formulations.     

临床病原菌的耐药性研究

目的:研究我院住院患者中分离的病原菌的耐药状况。方法:将2005年1月至2006年12月住院患者的血液、尿液、痰、生殖道分泌物、粪便以及伤口分泌物标本中分离的病原菌进行药敏试验和耐药率分析。结果:分离菌株主要为大肠埃希菌(17.6%)、肺炎克雷伯菌(16.1%)、铜绿假单胞菌(23.5%)、鲍曼不动杆菌(12.8%)、阴沟肠杆菌(19.8%)和金黄色葡萄球菌(10.2%)。产广谱β-内酰胺酶(ESBL)菌在肺炎克雷伯菌和大肠埃希菌中的检出率为41.3%。革兰阴性菌对哌拉西林/他唑巴坦、亚胺培南和美罗培南较为敏感,金黄色葡萄球菌对头孢菌素的耐药率超过70%。产ESBL菌对21种常用抗菌药的平均耐药率为74.1%。病原菌的检出率如下:产ESBL菌41.3%,金黄色葡萄球菌10.2%,耐苯唑西林金黄色葡萄球菌49.0%。未检出耐万古霉素葡萄球菌。结论:产ESBL菌和耐苯唑西林金黄色葡萄球菌有多重耐药性。病原菌分离株的药敏试验对临床选用有效抗菌药及其合理应用颇为重要。     

细菌RND外排泵的结构与作用机制研究进展

细菌RND(resistance-nodulation-cell division)外排泵是一类由内膜转运蛋白、周质融合蛋白、外膜外排蛋白组成的蛋白复合体,能够将抗菌药物和多种小分子化合物如毒素、染料、洗涤剂、脂质、群感信号分子等排出细菌细胞外。通过影响RND外排泵的组装和功能,可逆转多重耐药性的发生与防止耐药性的发展。细菌具有多种外排泵,本文以RND外排泵为代表,介绍与其结构以及外排药物的转运和调控机制的最新进展。     

多重耐药鲍曼不动杆菌临床分布及外排泵基因的检测

目的了解多重耐药鲍曼不动杆菌临床分布并检测其外排泵基因型。方法统计96株多重耐药鲍曼不动杆菌临床分布,用Kirby-Bauer法检测96株多重耐药鲍曼不动杆菌对15种抗菌药物的耐药情况,并用PCR扩增进行外排泵基因的检测。结果 96株多重耐药鲍曼不动杆菌主要分布在重症监护病房(54.2%)和呼吸内科(18.8%);对喹诺酮类、头孢菌素类、氨基糖苷类、四环素类抗菌药物的耐药率均在70%以上;分布在重症监护病房的52株多重耐药鲍曼不动杆菌中检测到ade B、ade R、ade S、ade J、ade E、abe M基因分别有18株(34.62%)、16株(30.77%)、18株(34.62%)、18株(34.62%)、0株、18株(34.62%);分布在呼吸内科的18株多重耐药鲍曼不动杆菌中检测到ade B、ade R、ade S、ade J、ade E、abe M基因分别有9株、8株、8株、8株、0株、8株。结论外排泵基因是分布在重症监护病房和呼吸内科的鲍曼不动杆菌发生多重耐药的重要因素。     

Saturable Process Involved in Active Efflux of Vincristine as a Mechanism of Multidrug Resistance in P388 Leukemia Cells

Kinetic analysis of vincristine (VCR) efflux in multidrug-resistant and parental P388 leukemia cells was performed to investigate the difference in activity between the two cell lines. Efflux velocities of VCR were directly determined from the slope of the initial release of drug induced by resuspending the preloaded cells in VCR-free medium, representing unidirectional efflux from intracellular free or loosely bound drug pools. Further, the equilibrium binding of VCR to whole-cell homogenates was analyzed by ultrafiltration to estimate intracellular unbound drug concentrations. A two-site binding model was found to fit the data best for both cell lines, and depletion of ATP by the addition of apyrase decreased binding. The binding parameters were similar between the two cell lines. A Hofstee plot of efflux demonstrated the existence of both linear and saturable transport of VCR in both cell lines. The greater maximum velocity observed with VCR efflux in the resistant cells suggests that an increased number of transporters causes greater activity of this process in the resistant cells.     

左氧氟沙星和美洛培南体外诱导铜绿假单胞菌耐药的外排机制研究

目的:研究左氧氟沙星和美洛培南对铜绿假单胞菌耐药的诱导作用及诱导耐药的外排机制。方法:多步诱导法对3株铜绿假单胞菌进行诱导耐药试验,对诱导出的菌株用微量肉汤稀释法测定加与不加外排泵抑制剂(苯丙氨酸-精氨酸-!萘酰胺)的最小抑菌浓度(MIC)的变化,PCR法检测外排泵基因oprM和mexB,并扩增mexR基因进行DNA测序。结果:3株菌均诱导出稳定的耐左氧氟沙星和美洛培南的耐药株,MIC值与原菌比较分别增加了16倍～64倍和2倍～16倍,加外排泵抑制剂后左氧氟沙星的MIC值降低了4倍～8倍;3株菌诱导前后均扩增出外排泵基因oprM和mexB,mexR测序结果与GenBankU23763比较,核苷酸序列第222位(C→T),氨基酸序列(CTG→TTG)均为LEU,为无义突变,核苷酸序列第436位插入1bp(A)移码框。结论:左氧氟沙星和美洛培南可诱导铜绿假单胞菌产生获得性耐药,其耐药机制与主动外排相关。     

细菌多重耐药外排泵抑制剂研究进展

细菌耐药性，尤其是多重耐药性（multi-drug resistance，MDR）已经成为非常严重的医疗问题，而多种类型细菌外排泵（efflux pumps）的存在是细菌多重耐药的重要机制，因此寻找有应用前景的外排泵抑制剂（efflux pump inhibitors，EPI）是十分必要且迫切的。目前已经发现外排泵抑制剂的作用机制分为：（1）干扰外排泵组装；（2）阻断外排泵能量来源；（3）阻碍底物通过外排通道；（4）机制未知。本文按照作用机制对已经发现的细菌多重耐药外排泵抑制剂的特点进行分述。     

Molecular evidence and functional expression of a novel drug efflux pump (ABCC2) in human corneal epithelium and rabbit cornea and its role in ocular drug efflux

Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium, thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the P-glycoprotein (P-gp) in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multidrug resistance associated proteins (MRPs) contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40-HCEC and rPCEC were selected for in vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [³H]-cyclosporine-A and [¹⁴C]-erythromycin, which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at ∼272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC, and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band ∼181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at ∼190 kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [³H]-cyclosporine-A and [¹⁴C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40-HCEC and rPCEC. Similarly a significant elevation in (A → B) permeability of [³H]-cyclosporine-A and [¹⁴C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A → B transport of [³H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding.     

Mechanisms of drug resistance in epilepsy: relevance for antiepileptic drug discovery

Background: Epilepsy, the most common chronic neurological pathology, is symptomatically treated by present antiepileptic drugs (AEDs) in about two-thirds of the cases. Unfortunately, this proportion has not been significantly reduced despite the introduction of several new-generation AEDs. Objective: This review challenges the utility of the paradigm of the excitation–inhibition imbalance for AED discovery and review mechanisms, presumed to be involved in drug-resistant epilepsy, with the purpose of discussing their relevance as targets for future AED discovery. Conclusion: Considering epilepsy as a mere imbalance between excitation and inhibition seems incapable of providing any proper basis for enabling future AED discovery to combat drug-resistant epilepsy as it oversimplifies a complex pathology, yet insufficiently understood. Two current hypotheses on the mechanisms of drug resistance in epilepsy highlight the roles of increased activity of blood–brain barrier multidrug transporter proteins and of alterations in the drug targets rendering them drug-insensitive. Both mechanisms are relevant but seem insufficient to account for the complexity of brain changes involved in drug-resistant epilepsy. Recent studies of drug-resistant epilepsy have revealed the involvement of inflammation processes, functional glia changes and altered intercellular communication related to gap junctions. This provides further, albeit not exhaustive, examples of targets to consider for future AED discovery. A successful strategy aimed at overcoming resistance to AEDs necessitates an integrated vision encompassing the basic features of intractable epilepsies.     

4‐Hydroxy‐α‐Tetralone and its Derivative as Drug Resistance Reversal Agents in Multi Drug Resistant Escherichia coli

The purpose of present investigation was to understand the drug resistance reversal mechanism of 4‐hydroxy‐α‐tetralone ( 1 ) isolated from Ammannia spp. along with its semi‐synthetic derivatives ( 1a – 1e ) using multidrug resistant Escherichia coli (MDREC). The test compounds did not show significant antibacterial activity of their own, but in combination, they reduced the minimum inhibitory concentration (MIC) of tetracycline (TET). In time kill assay, compound 1 and its derivative 1e in combination with TET reduced the cell viability in concentration dependent manner. Compounds 1 and 1e were also able to reduce the mutation prevention concentration of TET. Both compounds showed inhibition of ATP dependent efflux pumps. In real time polymerase chain reaction (RT‐PCR) study, compounds 1 and 1e alone and in combination with TET showed significant down expression of efflux pump gene (yojI) encoding multidrug ATP binding cassettes (ABC) transporter protein. Molecular mechanism was also supported by the in silico docking studies, which revealed significant binding affinity of compounds 1 and 1e with YojI. This study confirms that compound 1 and its derivative 1e are ABC efflux pump inhibitors which may be the basis for development of antibacterial combinations for the management of MDR infections from inexpensive natural product.