Discovery and Biological Characterization of the Auromomycin Chromophore as an Inhibitor of Biofilm Formation in Vibrio cholerae

Bacterial biofilms pose a significant challenge in clinical environments due to their inherent lack of susceptibility to antibiotic treatment. It is widely recognized that most pathogenic bacterial strains in the clinical setting persist in the biofilm state, and are the root cause of many recrudescent infections. The discovery and development of compounds capable of either inhibiting biofilm formation or initiating biofilm dispersal might provide new therapeutic avenues for reducing the number of hospital‐acquired, biofilm‐mediated infections. We detail here the application of our recently reported image‐based, high‐throughput screen to the discovery of microbially derived natural products with inhibitory activity against Vibrio cholerae biofilm. Examination of a prefractionated library of microbially derived marine natural products has led to the identification of a new biofilm inhibitor that is structurally unrelated to previously reported inhibitors and is one of the most potent inhibitors of V. cholerae reported to date. Combination of this compound with sub‐MIC concentrations of a number of clinically relevant antibiotics was shown to improve the inhibitory efficacy of this new compound compared to monotherapy treatments, and provides evidence for the potential therapeutic benefit of biofilm inhibitors in treating persistent biofilm‐mediated infections.     

Substrate Fragmentation for the Design of M. tuberculosis CYP121 Inhibitors

The cyclo‐dipeptide substrates of the essential M. tuberculosis (Mtb) enzyme CYP121 were deconstructed into their component fragments and screened against the enzyme. A number of hits were identified, one of which exhibited an unexpected inhibitor‐like binding mode. The inhibitory pharmacophore was elucidated, and fragment binding affinity was rapidly improved by synthetic elaboration guided by the structures of CYP121 substrates. The resulting inhibitors have low micromolar affinity, good predicted physicochemical properties and selectivity for CYP121 over other Mtb P450s. Spectroscopic characterisation of the inhibitors′ binding mode provides insight into the effect of weak nitrogen‐donor ligands on the P450 heme, an improved understanding of factors governing CYP121–ligand recognition and speculation into the biological role of the enzyme for Mtb.     

Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein‐Detected NMR Spectroscopy

An academic chemical screening approach was developed by using 2D protein‐detected NMR, and a 352‐chemical fragment library was screened against three different protein targets. The approach was optimized against two protein targets with known ligands: CXCL12 and BRD4. Principal component analysis reliably identified compounds that induced nonspecific NMR crosspeak broadening but did not unambiguously identify ligands with specific affinity (hits). For improved hit detection, a novel scoring metric—difference intensity analysis (DIA)—was devised that sums all positive and negative intensities from 2D difference spectra. Applying DIA quickly discriminated potential ligands from compounds inducing nonspecific NMR crosspeak broadening and other nonspecific effects. Subsequent NMR titrations validated chemotypes important for binding to CXCL12 and BRD4. A novel target, mitochondrial fission protein Fis1, was screened, and six hits were identified by using DIA. Screening these diverse protein targets identified quinones and catechols that induced nonspecific NMR crosspeak broadening, hampering NMR analyses, but are currently not computationally identified as pan‐assay interference compounds. The results established a streamlined screening workflow that can easily be scaled and adapted as part of a larger screening pipeline to identify fragment hits and assess relative binding affinities in the range of 0.3–1.6 mm . DIA could prove useful in library screening and other applications in which NMR chemical shift perturbations are measured.     

In Silico Evaluation of the Binding Site of Fucosyltransferase 8 and First Attempts to Synthesize an Inhibitor with Drug-Like Properties

Core fucosylation of N-glycans is catalyzed by fucosyltransferase 8 and is associated with various types of cancer. Most reported fucosyltransferase inhibitors contain non-drug-like features, such as charged groups. New starting points for the development of inhibitors of fucosyltransferase 8 using a fragment-based strategy are presented. Firstly, we discuss the potential of a new putative binding site of fucosyltransferase 8 that, according to a molecular dynamics (MD) simulation, is made accessible by a significant motion of the SH3 domain. This might enable the design of completely new inhibitor types for fucosyltransferase 8. Secondly, we have performed a docking study targeting the donor binding site of fucosyltransferase 8, and this yielded two fragments that were linked and trimmed in silico. The resulting ligand was synthesized. Saturation transfer difference (STD) NMR confirmed binding of the ligand featuring a pyrazole core that mimics the guanine moiety. This ligand represents the first low-molecular-weight compound for the development of inhibitors of fucosyltransferase 8 with drug-like properties.     

Fragment Growing to Design Optimized Inhibitors for Human Blood Group B Galactosyltransferase (GTB)

Human blood group B galactosyltransferase (GTB) catalyzes the galactosylation of the H antigen and is responsible for the formation of the blood group antigen of phenotype B. The ABO blood group system is well studied and routinely serotyped before transfusion and transplantation. Blood type subgroups have been repeatedly linked to an increased occurrence of diseases (e.g., a highly increased incidence rate for pancreatic cancer for individuals with blood group phenotype B). 3-Phenyl-5-(piperazin-1-yl)-1,2,4-thiadiazole 1 has previously been described to inhibit GTB with a K_i value of 800 μm . In this work, we describe a computer-guided fragment-growing approach for the optimization of this fragment that was subsequently realized by synthesizing the most promising ligands. Enlarging the phenyl moiety of fragment 1 to a naphthyl moiety resulted in ligand 3-(naphthalene-1-yl)-5-(piperazin-1-yl)-1,2,4-thiadiazole 2 a , which shows a threefold improvement in binding affinity (K_i=271 μm ).     

Virtual Fragment Screening Identification of a Quinoline‐5,8‐dicarboxylic Acid Derivative as a Selective JMJD3 Inhibitor

The quinoline‐5,8 dicarboxylic acid scaffold has been identified by a fragment‐based approach as new potential lead compound for the development of JMJD3 inhibitors. Among them, 3‐(2,4‐dimethoxypyrimidin‐5‐yl)quinoline‐5,8‐dicarboxylic acid (compound 3 ) shows low micromolar inhibitory activity against Jumonji domain‐containing protein 3 (JMJD3). The experimental evaluation of inhibitory activity against seven related isoforms of JMJD3 highlighted an unprecedented selectivity toward the biological target of interest.     

抗霍乱弧菌O1群单克隆抗体的制备及初步鉴定

目的制备抗霍乱弧菌O1群特异性单克隆抗体(McAb),并进行鉴定。方法以福尔马林灭活的O1群霍乱弧菌免疫BALB/c小鼠,应用细胞融合技术建立分泌抗O1群霍乱弧菌McAb的杂交瘤细胞株。对配对较好的6个细胞株分泌的McAb进行腹水效价、免疫球蛋白类及亚类、抗体亲和常数测定及凝集试验和稳定性试验。与细菌培养法对比检测208份临床标本。结果6个细胞株分泌的McAb腹水效价均为105,均为IgM;抗体亲和常数为1.84×105～5.38×107;且均与O1群霍乱弧菌菌株发生凝集反应,与霍乱弧菌O139群、大肠杆菌、出血性大肠杆菌O157∶H7、副溶血弧菌、麦弧菌、河弧菌、结肠炎耶尔森菌、普通变形杆菌、痢疾杆菌、伤寒杆菌及各型副伤寒杆菌均不发生凝集反应。6个分泌McAb的细胞株连续培养15周,经液氮冻存12个月后复苏,仍能稳定分泌抗体;与细菌培养法对比检测208份临床标本,特异性和灵敏度均达100%。结论制备的McAb具有较高的特异性,有可能用于开发霍乱弧菌O1群的检测试剂。     

霍乱弧菌外膜蛋白抗原的共同性和特异性

通过免疫吸收试验证明,不同生物型和血清型的霍乱弧菌外膜蛋白(OMP)是共同抗原,它们之间可互相完全吸收,共同抗原是分子量约为47K和86K的IA组分。OMP和IA组分对01霍乱弧菌是特异性抗原,其抗IA血清经LPS吸收只能与霍乱弧菌发生阳性反应,与NAG和其它革兰氏阴性菌的参试株不发生阳性反应。霍乱弧菌LPS是与革兰氏阴性菌发生交叉反应的物质,以LPS为基础制备的多价血清及其分型血清,难免发生非特异的交叉反应。