新型甲氧苄啶耐药二氢叶酸还原酶DfrB7的生化分析与宿主菌耐药机制研究

【目的】分析新型甲氧苄啶获得性耐药蛋白DfrB7的生化性质,探究其与B家族代表性的二氢叶酸还原酶(DHFR)对甲氧苄啶获得性耐药的生化基础。【方法】构建系统进化树分析B家族DHFRs与新型DfrB7的进化关系。将dfr基因分别构建到pACYC184和pET15b(+)载体并转化到相应大肠杆菌中。使用微量肉汤稀释法确定克隆菌株对甲氧苄啶的耐药性。测定DHFRs使用NADPH作为质子供体催化二氢叶酸还原的酶活反应参数。通过等温滴定量热法测定甲氧苄啶的解离常数。【结果】克隆到大肠杆菌中的新型dfrB7基因表现出对甲氧苄啶的高耐药表型。系统进化树确定了dfrB7编码B家族的DHFRs。检测并比较DfrB7和代表性DHFRs的酶活性质、抑制剂的亲和力,与染色体上DHFR相比,DfrB7与DfrB1均表现出显著降低的催化活性,通过生化实验证实B家族二氢叶酸还原酶对甲氧苄啶结合力极差。【结论】新型dfrB7基因编码的DfrB7具有B家族二氢叶酸还原酶的普遍特征。B家族二氢叶酸还原酶赋予宿主菌对甲氧苄啶的获得性耐药与该酶对甲氧苄啶的低亲和力有关。     

Development of novel isatin–nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing–bacteria

Joining the global fight against Tuberculosis, the world''s most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (5a–m and 9a–c) as promising anti-tubercular and antibacterial agents. The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49–7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.     

Development of a novel,multifunctional, membrane-interactive pyridinium salt with potent anticancer activity

The synthesis and biological evaluation of a novel pyridinium salt is reported. Initial membrane interaction with isolated phospholipid monolayers was obtained with the pyridinium salt, and two neutral analogues for comparison, and the anticancer effects of the best compound established using a cytotoxicity screening assay against glioma cells using both an established cell line and three short-term cell cultures—one of which has been largely resistant to all chemotherapeutic drugs tested to date. The results indicate that the pyridinium salt exhibits potent anticancer activity (EC₅₀s = 9.8–312.5 μM) on all cell types, including the resistant one, for a continuous treatment of 72 h. Microscopic examination of the treated cells using a trypan blue exclusion assay showed membrane lysis had occurred. Therefore, this letter highlights the potential for a new class of pyridinium salt to be developed as a much needed alternative treatment for glioma chemotherapy.     

CP5484, a novel quaternary carbapenem with potent anti-MRSA activity and reduced toxicity

A new series of 1beta-methyl carbapenems possessing a 6,7-disubstituted imidazo[5,1-b]thiazol-2-yl group directly attached to the C-2 position of the carbapenem nucleus was prepared, and the activities of these compounds against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. To study the effect of basic moieties on anti-MRSA activity, we introduced an amino, or imino, or amidino group at the 6-position of imidazo[5,1-b]thiazole in place of the carbamoylmethyl moiety of CP5068. Anti-MRSA activities of almost all basic group-substituted carbapenems were improved, though some of the compounds showed stronger acute toxicity in mice than IPM. In order to decrease the toxicity without decreasing the activity, we introduced various additional functionalities around the basic moiety. Finally, we obtained CP5484, which has excellent anti-MRSA activity and low acute toxicity.     

Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Isolation of a novel basic FGF-binding peptide with potent antiangiogenetic activity

Basic fibroblast growth factor (bFGF), which plays an important role in tumour angiogenesis and progression, provides a potential target for cancer therapy. Here we screened a phage display heptapeptide library with bFGF and identified 11 specific bFGF-binding phage clones. Two of these clones had identical sequence and the corresponding peptide (referred to as P7) showed high homology to the immunoglobulin-like (Ig-like) domain III (D3) of high-affinity bFGF receptors, FGFR1 (IIIc) and FGFR2 (IIIc). The P7 peptide and its corresponding motif in D3 of FGFRs both carried negative charges and shared similar hydrophobic profiles. Functional analysis demonstrated that synthetic P7 peptides mediate strong inhibition of bFGF-induced cell proliferation and neovascularization. Our results demonstrate that the P7 peptide is a potent bFGF antagonist with strong antiangiogenetic activity, and might have therapeutic potential in cancer therapy.     

A novel artemisinin-quinine hybrid with potent antimalarial activity

Artemisinin was reduced to dihydroartemisinin and coupled to a carboxylic acid derivative of quinine via an ester linkage. This novel hybrid molecule had potent activity against the 3D7 and (drug-resistant) FcB1 strains of Plasmodium falciparum in culture. The activity was superior to that of artemisinin alone, quinine alone, or a 1:1 mixture of artemisinin and quinine.     

Identification of a novel boronic acid as a potent,selective, and orally active hormone sensitive lipase inhibitor

Hormone sensitive lipase (HSL) is an attractive therapeutic target of dyslipidemia. We designed and synthesized several compounds as reversible HSL inhibitors with a focus on hydrophobic interactions, which was thought to be effective upon the HSL inhibitory activity. In these efforts, we identified boronated compound 12 showing a potent HSL inhibitory activity with an IC₅₀ value of 7 nM and a high selectivity against cholinesterases. Furthermore, compound 12 is the first boron containing HSL inhibitor that has shown an antilipolytic effect in rats after oral administration at 3 mg/kg.     

Cathepsin V, a novel and potent elastolytic activity expressed in activated macrophages

Atherosclerosis is characterized by a thickening and loss of elasticity of the arterial wall. Loss of elasticity has been attributed to the degradation of the arterial elastin matrix. Cathepsins K and S are papain-like cysteine proteases with known elastolytic activities, and both enzymes have been identified in macrophages present in plaque areas of diseased blood vessels. Here we demonstrate that macrophages express a third elastolytic cysteine protease, cathepsin V, which exhibits the most potent elastase activity yet described among human proteases and that cathepsin V is present in atherosclerotic plaque specimens. Approximately 60% of the total elastolytic activity of macrophages can be attributed to cysteine proteases with cathepsins V, K, and S contributing equally. From this 60%, two-thirds occur extracellularly and one-third intracellularly with the latter credited to cathepsin V. Ubiquitously expressed glycosaminoglycans (GAGs) such as chondroitin sulfate specifically inhibit the elastolytic activities of cathepsins V and K via the formation of specific cathepsin-GAG complexes. In contrast, cathepsin S, which does not form complexes with chondroitin sulfate is not inhibited; thus suggesting a specific regulation of elastolytic activities of cathepsins by GAGs. Because the GAG content is reduced in atherosclerotic plaques, an increase of cathepsins V and K activities may accelerate the destruction of the elastin matrix in diseased arteries.     

Design,synthesis, and biological activity of novel tetrahydropyrazolopyridone derivatives as FXa inhibitors with potent anticoagulant activity

A series of novel tetrahydropyrazolopyridone derivatives containing 1,3,4-triazole, triazolylmethyl, and partially saturated heterocyclic moieties as P2 binding element was designed, synthesized, and evaluated in vitro for anticoagulant activity in human and rabbit plasma. All compounds showed moderate to significant potency, and compounds 15b, 15c, 20b, 20c, and 22b were further examined for their inhibitory activity against human FXa in vitro. While compounds 15c and 22b were tested for rat venous thrombosis in vivo. The most promising compound 15c, with an IC₅₀ (FXa) value of 0.14 μM and 98% inhibition rate, warranted further investigation as an FXa inhibitor.     

Synthesis and antifungal activity of ASP9726, a novel echinocandin with potent Aspergillus hyphal growth inhibition

The synthesis and antifungal activity of ASP9726, a novel echinocandin with potent Aspergillus hyphal growth inhibition and significantly improved MIC against Candida parapsilosis and echinocandin resistant-Candida is described.     

Design,synthesis and potent cytotoxic activity of novel podophyllotoxin derivatives

Twenty new acyl thiourea derivatives of podophyllotoxin and 4′-demethylepipodophyllotoxin were prepared and screened for their cytotoxicity against four human tumor cell lines, A-549, DU-145, KB, and KBvin. With IC₅₀ values of 0.098–1.13 μM, compounds 13b, 13c, and 13o displayed much better cytotoxic activity than the control etoposide. Most importantly, 13b and 13o exhibited promising cytotoxicity against the drug resistant tumor cell line KBvin with IC₅₀ values of 0.098 and 0.13 μM, respectively, while etoposide lost activity completely. Structure–activity relationship (SAR) correlations of the new derivatives have been established. Compounds 13b and 13o merit further development as a new generation of epipodophyllotoxin-derived antitumor clinical trial candidates.     

Eryloside F, a novel penasterol disaccharide possessing potent thrombin receptor antagonist activity

We report the discovery of Eryloside F, a novel disaccharide of the steroidal carboxylic acid penasterol, isolated from an extract of the marine sponge Erylus formosus. The compound is a potent thrombin receptor antagonist, and furthermore inhibits human platelet aggregation in vitro.     

Synthesis of novel di- and tricationic carbapenems with potent anti-MRSA activity

A new series of 1β-methyl carbapenems possessing a 6,7-disubstituted imidazo[5,1-b]thiazol-2-yl group directly attached to the C-2 position of the carbapenem nucleus was prepared, and their activities against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. First, a benzyl moiety was introduced at the C-6 position of imidazo[5,1-b]thiazole attached to the carbapenem. These benzylated molecules showed potent anti-MRSA activity, but poor water solubility. In order to overcome this drawback, we designed and synthesized di- and tricationic carbapenems and finally discovered a novel carbapenem (15i), which exhibited excellent anti-MRSA activity and good water solubility.