Design and Experimental Evaluation of a Peptide Antagonist against Amyloid β(1–42) Interactions with Calmodulin and Calbindin-D28k

Amyloid β_1–42 (Aβ(1–42)) oligomers have been linked to the pathogenesis of Alzheimer’s disease (AD). Intracellular calcium (Ca²⁺) homeostasis dysregulation with subsequent alterations of neuronal excitability has been proposed to mediate Aβ neurotoxicity in AD. The Ca²⁺ binding proteins calmodulin (CaM) and calbindin-D28k, whose expression levels are lowered in human AD brains, have relevant roles in neuronal survival and activity. In previous works, we have shown that CaM has a high affinity for Aβ(1–42) oligomers and extensively binds internalized Aβ(1–42) in neurons. In this work, we have designed a hydrophobic peptide of 10 amino acid residues: VFAFAMAFML (amidated-C-terminus amino acid) mimicking the interacting domain of CaM with Aβ (1–42), using a combined strategy based on the experimental results obtained for Aβ(1–42) binding to CaM and in silico docking analysis. The increase in the fluorescence intensity of Aβ(1–42) HiLyte^TM-Fluor555 has been used to monitor the kinetics of complex formation with CaM and with calbindin-D28k. The complexation between nanomolar concentrations of Aβ(1–42) and calbindin-D28k is also a novel finding reported in this work. We found that the synthetic peptide VFAFAMAFML (amidated-C-terminus amino acid) is a potent inhibitor of the formation of Aβ(1–42):CaM and of Aβ(1–42):calbindin-D28k complexes.     

Structure‐Guided Rational Design of α/β‐Peptide Foldamers with High Affinity for BCL‐2 Family Prosurvival Proteins

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β‐peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl‐x_L but weakly to Mcl‐1. The crystal structure of the Puma‐derived α/β‐peptide complexed to Bcl‐x_L was used as the basis for computational design of variants intended to display improved binding to Mcl‐1. Molecular modelling suggested modification of three α residues of the original α/β backbone. Individually, each substitution caused only a modest (4‐ to 15‐fold) gain in affinity; however, together the three substitutions led to a 250‐fold increase in binding to Mcl‐1. These modifications had very little effect on affinity for Bcl‐x_L. Crystal structures of a number of the new α/β‐peptides bound to either Mcl‐1 or Bcl‐x_L validated the selection of each substitution. Overall, our findings demonstrate that structure‐guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.     

抗菌肽的结构功能关系及分子设计的研究现状

抗菌肽是一种广泛存于自然界中具有广谱抗菌作用的小分子多肽。文章首先对抗菌肽的结构进行分类、从肽链长度、α-螺旋结构、疏水性、两亲性和净电荷5个方面分析了分子结构对活性的影响、并对抗菌肽的分子设计进行了综述,对其发展前景进行了展望。     

HTPP-MDC原核表达载体的构建及在大肠杆菌中的表达

通过双酶切、连接、转化等方法将肝靶向穿膜肽-家蝇天蚕素（HTPP-MDC）融合基因克隆至原核表达载体pET32a（＋）上,构建重组表达质粒HTPP-MDC/pET32a。重组质粒转化E.coli BL21（DE3）后,以IPTG诱导,SDS-PAGE分析表明重组融合蛋白得到了可溶性表达,Western blot杂交证实了表达蛋白的抗原活性。为HTPP-MDC的生物学活性研究及产业化开发奠定了基础。     

The X-ray structure of (MeBm2t)1-cyclosporin complexed with cyclophilin A provides an explanation for its anomalously high immunosuppressive activity

For most of the cyclosporin A (CsA) analogs, there is generallya good correlation between cyclophilin binding and immunosuppression.However, this relationship does not seem to hold for 4-[(E)-2-butenyl]-4,4,N-trimethyl-L-threonine¹(MeBm₂t)¹-CsA.Its affinity for cyclophilin was reported to be {small tilde}1percent; that of CsA and its immunosuppressive activity invitro was shown to be {small tilde} 30% that of CsA. We reporthere the crystal structure of a complex between recombinanthuman cyclophilin A (CypA) and (MeBm₂t)¹-CsA which has beendetermined by X-ray crystallography at 2.2 Å resolutionand refined to an Rfactor of 16.3%. (MeBm₂t)¹-CsA shows a similarbound conformation and network of interactions to CypA as CsA.The measured lower affinity for CypA cannot therefore be explainedby a different mode of binding. We propose that the poor affinityto CypA could be accounted for by the existence of an equilibriumin aqueous solution between a ‘cyclophilin bound conformation’and a ‘nonbinding conformation’ of (MeBm₂t)¹-CsA.The relatively high immunosuppressive activity is suggestedto result from slight conformational differences observed inthe effector domain     

Synthesis, solution structure and immune recognition of an epidermal growth factor-like domain from Plasmodium falciparum merozoite surface protein-1

The Plasmodium falciparum merozoite surface protein-1 19 kDa fragment (MSP-1(19)) comprises two closely packed EGF-like domains (EGF=epidermal growth factor), each stabilized by three disulfide bonds. The native conformation of this protein is important for eliciting P. falciparum growth inhibitory antibodies. Here we show that the N-terminal EGF domain alone can be chemically synthesized and efficiently refolded to a native-like state, as shown by its solution structure as determined by NMR spectroscopy. In order to study its immunogenicity, the domain was coupled through its N terminus to a phospholipid and incorporated into reconstituted influenza virus-like particles (virosomes). When used to immunize mice, the peptide-loaded virosomes elicited potent humoral immune responses that were shown by Western blots and immunofluorescence assays to cross-react with native MSP-1 on the surfaces of P. falciparum blood stage parasites. This opens the way for a medicinal chemistry-oriented approach to the study and optimization of the antigenicity of the protein as a potential malaria vaccine candidate, whilst exploiting the immunopotentiating properties of influenza virosomes as a delivery vehicle.     

Fusarin C biosynthesis in Fusarium moniliforme and Fusarium venenatum

Fragments of polyketide synthase (PKS) genes were amplified from complementary DNA (cDNA) of the fusarin C producing filamentous fungi Fusarium moniliforme and Fusarium venenatum by using degenerate oligonucleotides designed to select for fungal PKS C-methyltransferase (CMeT) domains. The PCR products, which were highly homologous to fragments of known fungal PKS CMeT domains, were used to probe cDNA and genomic DNA (gDNA) libraries of F. moniliforme and F. venenatum. A 4.0 kb cDNA clone from F. venenatum was isolated and used to prepare a hygromycin-resistance knockout cassette, which was used to produce a fusarin-deficient strain of F. venenatum (kb = 1000 bp). Similarly, a 26 kb genomic fragment, isolated on two overlapping clones from F. moniliforme, encoded a complete iterative Type I PKS fused to an unusual nonribosomal peptide synthase module. Once again, targeted gene disruption produced a fusarin-deficient strain, thereby proving that this synthase is responsible for the first steps of fusarin biosynthesis.     

Synthesis and Application of Peptide Arrays: Quo Vadis SPOT Technology

Hitting the SPOT : In 1992, Ronald Frank published the first seminal paper on simultaneous parallel synthesis of multiple peptides on filter paper. He defined the approach as SPOT synthesis, an easy technique for positionally addressable, parallel chemical synthesis on a membrane support. Here, a basic overview of this technology is presented and a recently published applications are highlighted. At the end, the future of peptide arrays is discussed.

     

Cover Picture: Structure–Activity Relationship Analysis of the Peptide Deformylase Inhibitor 5‐Bromo‐1H‐indole‐3‐acetohydroxamic Acid (ChemMedChem 2/2009)

The cover picture shows a “reverse” indole derivative in complex with Bacillus stearothermophilus peptide deformylase (PDF). This compound was selected from a structure–activity relationship study as a potent inhibitor of bacterial PDFs and shows antibacterial activity toward Bacillus subtilis as well as other pathogens such as Streptococcus pneumoniae and Staphylococcus aureus. For more details, see the Full Paper by I. Artaud et al. on p. 261 ff.

     

Salen-Mn(Ⅲ)和Salen-Mo(Ⅳ)配合物在介孔硅胶上接枝及烯烃环氧化催化性能

通过肽键作用将Salen型金属配合物接枝到介孔硅胶孔道中生成固相催化剂,采用红外光谱、热重分析和元素分析等对制备的固相催化剂进行表征,结果证实,Salen型金属配合物成功接枝到介孔硅胶载体上。以环辛烯和环己烯为反应底物,叔丁基过氧化氢和过氧化氢为氧化剂,比较均相Salen型催化剂和多相Salen型催化剂的催化活性。制备的均相Salen型催化剂和利用肽键键合制备的固相催化剂均具有一定的催化性能,Mo-Salen催化活性更高,是因为叔丁基过氧化氢在Mo-Salen存在下易分解。固相催化剂活性≤75℃时稳定,在氧化剂叔丁基过氧化氢和过氧化氢作用下具有较好的稳定性能。重复实验中,金属离子流失量很小,催化活性和TOF值未降低,表明利用肽键制备的固相催化剂催化活性稳定,为固相催化剂的制备开辟新思路。