Milbemycins的结构修饰

综述了一大类重要的天然产物 Milbemycins在近十年来的研究进展情况。并重点对此类化合物的结构化学修饰方法进行了论述 。

Mining and fine-tuning sugar uptake system for titer improvement of milbemycins in Streptomyces bingchenggensis

Dramatic decrease of sugar uptake is a general phenomenon in Streptomyces at stationary phase,when antibiotics are extensively produced.Milbemycins produced by Streptomyces bingchenggensis are a group of valuable macrolide biopesticides,while the low yield and titer impede their broad applications in agricultural field.Considering that inadequate sugar uptake generally hinders titer improvement of desired products,we mined the underlying sugar uptake systems and fine-tuned their expression in this work.First,we screened the candidates at both genomic and transcriptomic level in S.bingchenggensis.Then,two ATP-binding cassette transporters named TP2 and TP5 were characterized to improve milbemycin titer and yield significantly.Next,the appropriate native temporal promoters were selected and used to tune the expression of TP2 and TP5,resulting in a maximal milbemycin A3/A4 titer increase by 36.9%to 3321 mg/L.Finally,TP2 and TP5 were broadly finetuned in another two macrolide biopesticide producers Streptomyces avermitilis and Streptomyces cyaneogriseus,leading to a maximal titer improvement of 34.1%and 52.6%for avermectin B1a and nemadectin,respectively.This work provides useful transporter tools and corresponding engineering strategy for Streptomyces.

Milbemycin Oxime半抗原设计及其抗体制备

通过milbemycin oxime A4、A3(MILO)肟羟基与琥珀酸酐反应,合成了半抗原MILO-5-琥珀酰半酯,采用碳化二亚胺法将其与载体蛋白BSA偶联制备人工抗原.以此人工抗原免疫新西兰大白兔获得多克隆抗体,抗体效价达1:12 800;用此抗体建立的间接竞争酶联免疫吸附(CI-ELISA)检测方法,线性范围在16.00～4 394.18 ng/mL,最低检测限(I10)为6.28 ng/mL.

Pharmacokinetics of Milbemycin Oxime in Dogs Following Its Intravenous and Oral Administration

The pharmacokinetics of milbemycin oxime was investigated in dogs following oral(per os, PO) and intravenous(IV) administration. Three groups of dogs received milbemycin oxime tablets as a single PO dose equal to 0.25, 0.5 and 1.0 mg · kg-1 of milbemycin oxime, respectively, another group received a single IV dose of 0.5 mg · kg-1. Blood samples were collected at predetermined times after drug administration and the milbemycin oxime concentrations in plasma were determined by LC-MS/MS. The drug protein binding in dog plasma in vitro was determined by equilibrium dialysis at concentrations spanning the range of values observed in vivo in dog plasma. After PO administration at doses of 0.25, 0.5 and 1.0 mg · kg-1, milbemycin oxime was slowly absorbed and eliminated, the time to reach the maximum plasma concentration(Tmax) was 4.14±0.20, 4.27±0.14 and 4.06±0.13 h, the mean absorption time(MAT) was 19.06, 13.67 and 11.77 h, the terminal rate half-life(t1/2λz) was 15.06±0.37, 11.09±0.54 and 9.76±0.89 h and the total body clearance(Cl) was 1.15±0.05, 1.18±0.03 and 1.17±0.07 m L · min-1 · kg-1, respectively. The maximum plasma concentration(Cmax, 36.50±1.40, 76.11±2.77 and 182.05±7.20 ng · m L-1, respectively) and the area under the first-moment curve(AUC-10→∞, 985.83±49.46, 1 663.12±51.42 and 3 558.04±197.88 mg · h · L, respectively) increased accordingly to the administered dose rates; the oral bioavailabilities were estimated to be 88.61%, 74.75% and 79.96%, respectively. The values of fu were 0.12%, 0.14% and 0.13% in dog plasma, respectively. In conclusion, the pharmacokinetics of milbemycin oxime in dogs following oral administration revealed its higher oral bioavailability and advantageous pharmacokinetic properties, such as its lower total body clearance and longer elimination half-life, and indicated that the single oral dose of 0.50 mg · kg-1 of milbemycin oxime which was recommended in all the parasitological efficacy studies allowed an adequate concentration of the drug.

Effect of Milbemycin oxime on Toxocara canis Eggs and Larvae

Toxocara canis(T.canis)is one of the most important zoonotic parasites of dogs.The aim of the present study was to perform an in vitro analysis of the effect of Milbemycin oxime on T.canis eggs following exposure to a concentration gradient of the drug and to determine the inflammatory reaction produced by the infective T.canis larvae in mice.The present study was undertaken using the model nematode,T.canis,to investigate the effect of Milbemycin oxime on T.canis eggs and larvae.T.canis eggs were exposed to a concentration gradient of Milbemycin oxime in vitro,the higher concentration of Milbemycin oxime was,the lower percentage of infective stage larvae was.Light micrographs showed that Milbemycin oxime induced eggs dissolved and eggshell broken.Histological analyses of mice that stained with hematoxylin and eosin(H&E)showed in lower dosing(10-7 and 10-8 g·mL-1)drug-treated groups,atrophy of alveolar space and interalveolar septum thickening appeared,inflammatory infiltrates accompanied with erythrocytes around blood vessels and bronchioles presented.In higher dosing(10-6,10-5 and 10-4 g·mL-1)drug-treated groups,low-grade or no pathological changes occurred,indicating that Milbemycin oxime could obviously decrease the inflammatory reaction produced by the infection of T.canis larvae in vivo.

Efficacy of Afoxolaner Alone or in Combination with Milbemycin Oxime against Rhipicephalus sanguineus Lato Sensu in Naturally Infested Dogs in Colombia

The brown dog tick (Rhipicephalus sanguineus lato sensu) is the tick that most affects dogs worldwide and is therefore the main blood pathogen vector in dogs. The efficacy of afoxolaner 2.7 to 7.1 mg/kg NexGard® (group A) and afoxolaner plus milbemycin oxime 2.5 to 5.4 mg/kg and 0.5 to 1.1 mg/kg respectively NexGard Spectra® (Group B) against R. sanguineus, was evaluated in naturally infected sheltered dogs under high challenging conditions in four different areas of Colombia (Antioquia, Córdoba, Santander, and Meta). Tick counts (alive, dead, attached, and unattached) were performed on treated dogs, the average was calculated for the different areas to evaluate the efficacy of each treatment at six different times post-treatment (24 h, 48 h, 7 d, 14 d, 21 d, 30 d). None of the dogs showed adverse events related to the treatments. The average tick number pre-treatment was 68 in group A and 78.3 for group B indicating a strong natural infection of the dogs and their environment. Efficacy after 24 h against R. sanguineus was always above 90% with ≥97.4% for NexGard® and ≥93.7% for NexGard Spectra®. Consistent results were observed along all the observation periods with final efficacies (day 30) of ≥99.8% and 98.3% for NexGard® and NexGard Spectra®, respectively. In conclusion, both NexGard® and Nexgard Spectra® provided a curative effect and sustained efficacy against Rhipicephalus sanguineus for at least 30 days in highly contaminated shelter environments.

Transcriptome-guided identification of a four-component system, SbrH1-R, that modulates milbemycin biosynthesis by influencing gene cluster expression, precursor supply, and antibiotic efflux

Streptomyces can produce numerous antibiotics and many other bioactive compounds.Recently,the molecular mechanisms of transcriptional regulators in control of antibiotic production by influencing the expression of biosynthetic gene clusters(BGCs)have been extensively studied.However,for regulators that affect both antibiotic production and cell growth,the way to influence antibiotic production may be diverse,but related studies are limited.Here,based on time-course transcriptome analysis,a four-component system,SbrH1-R,consisting of the two-component system SbrKR(SBI_03479/3478)and two hypothetical proteins SbrH1(SBI_03481)and SbrH2(SBI_03480)potentially related with the biosynthesis of milbemycins was identified in Streptomyces bingchenggensis BC-101-4.Deletion of sbrH1-R resulted in weakened cell growth but a 110%increase of milbemycin production compared with that in BC-101-4.Comparative transcriptome analyses of the sbrH1-R mutant and BC-101-4 revealed that SbrH1-R not only indirectly represses milbemycin BGC expression,but also inhibits milbemycin production by modulating expression levels of genes related to precursor supply and antibiotic efflux.Further genetic experiments identified several new targets,including five precursor supply-associated reactions/pathways(e.g.,the reaction from pyruvate to acetyl-CoA,the reaction from acetyl-CoA to citrate,the fatty acidβ-oxidation process,and the branched chain amino acid and phenylalanine acid degradation pathways)and a milbemycin exporter system(MilEX2)that can be engineered for milbemycin overproduction.These results shed new light on the understanding of regulation of milbemycin biosynthesis and provide useful targets for future metabolic engineering of the native host to improve milbemycin production.