Structure-based design,synthesis and biological testing of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI₅₀ in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.     

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ～50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.     

Molecular design of an acid–base cooperative catalyst for RNA cleavage based on a dizinc complex

The effects of donor groups of dizinc complexes, formed from a 2:1 mixture of Zn(II) and a dinucleating ligand, on adenylyl(3'-5')adenosine (ApA) cleavage have been studied. Two dinucleating ligands were used: one had two 2-pyridylmethyl and two 2-hydroxyethyl moieties on the 1,3-diamino-2-propanol linker moiety (2), and the other had two 2-pyridylmethyl and two carboxymethyl moieties on the 1,3-diamino-2-propanol linker moiety (3(2-)). The dizinc complex with2 [(Zn(2+))(2)-2] showed higher activities toward ApA cleavage than the dizinc complex using an analogous dinucleating ligand having four 2-pyridylmethyl donor moieties [(Zn(2+))(2)-1] at pH 5-8. The former showed a bell-shaped pH-rate constant profile, whereas the latter showed a sigmoidal pattern. The differences in the pH-rate constant profile are attributable to the various distributions of the monohydroxo-dizinc species, i.e. dideprotonated species, which are responsible for ApA cleavage. The monohydroxo species of (Zn(2+))(2)-2 has two acidic protons, which are not present in the corresponding monohydroxo species of (Zn(2+))(2)-1. The existence of both intracomplex acid (ROH or H(2)O) and base catalysts (RO(-) or OH(-)) in (Zn(2+))(2)-2 can explain its higher activity toward ApA cleavage than that of (Zn(2+))(2)-1. In contrast, (Zn(2+))(2)-3(2-) showed lower activity toward ApA cleavage at pH 7.0, which can be ascribed to the absence of the monohydroxo-dizinc species under these conditions.     

Kojic acid–amino acid conjugates as tyrosinase inhibitors

Kojic acid (KA), a well known tyrosinase inhibitor, has insufficient inhibitory activity and stability. We modified KA with amino acids and screened their tyrosinase inhibitory activity. Among them, kojic acid–phenylalanine amide (KA-F-NH₂) showed the strongest inhibitory activity, which was maintained for over 3 months at 50 °C, and acted as a noncompetitive inhibitor as determined by kinetic analysis. It also exhibited dopachrome reducing activity. We also propose a new tyrosinase inhibition mechanism based on the docking simulation data.     

Growth factor signaling to mTORC1 by amino acid–laden macropinosomes

The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid–dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow–derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficiently fast to explain growth factor–mediated signaling by amino acids. Therefore, the amino acid–laden macropinosome is an essential and discrete unit of growth factor receptor signaling to mTORC1.     

Mapping sites of positive selection and amino acid diversification in the HIV genome: an alternative approach to vaccine design?

A safe and effective HIV-1 vaccine is urgently needed to control the worldwide AIDS epidemic. Traditional methods of vaccine development have been frustratingly slow, and it is becoming increasingly apparent that radical new approaches may be required. Computational and mathematical approaches, combined with evolutionary reasoning, may provide new insights for the design of an efficacious AIDS vaccine. Here, we used codon-based substitution models and maximum-likelihood (ML) methods to identify positively selected sites that are likely to be involved in the immune control of HIV-1. Analysis of subtypes B and C revealed widespread adaptive evolution. Positively selected amino acids were detected in all nine HIV-1 proteins, including Env. Of particular interest was the high level of positive selection within the C-terminal regions of the immediate-early regulatory proteins, Tat and Rev. Many of the amino acid replacements were associated with the emergence of novel (or alternative) myristylation and casein kinase II (CKII) phosphorylation sites. The impact of these changes on the conformation and antigenicity of Tat and Rev remains to be established. In rhesus macaques, a single CTL-associated amino substitution in Tat has been linked to escape from acute SIV infection. Understanding the relationship between host-driven positive selection and antigenic variation may lead to the development of novel vaccine strategies that preempt the escape process.     

Synthesis of camptothecin–amino acid carbamate linkers

A more convenient and facile approach for the synthesis and production of camptothecin–amino acids carbamate linkers, that can be used in the synthesis of bioconjugate peptides JF-10-81, JF-10-71, and other peptide analogs designed to target somatostatin receptors has been described.     

An amino acid packing code for α-helical structure and protein design

This work demonstrates that all packing in α-helices can be simplified to repetitive patterns of a single motif: the knob-socket. Using the precision of Voronoi Polyhedra/Delauney Tessellations to identify contacts, the knob-socket is a four-residue tetrahedral motif: a knob residue on one α-helix packs into the three-residue socket on another α-helix. The principle of the knob-socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an α-helix, the three-residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob-socket motifs between two α-helices. Furthermore, the knob-socket model classifies three types of sockets: (1) free, favoring only intra-helical packing; (2) filled, favoring inter-helical interactions; and (3) non, disfavoring α-helical structure. The amino acid propensities in these three socket classes essentially represent an amino acid code for structure in α-helical packing. Using this code, we used a novel yet straightforward approach for the design of α-helical structure to validate the knob-socket model. Unique sequences for three peptides were created to produce a predicted amount of α-helical structure: mostly helical, some helical, and no helix. These three peptides were synthesized, and helical content was assessed using CD spectroscopy. The measured α-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob-socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure.     

Structure-based design of ester compounds to inhibit MLL complex catalytic activity by targeting mixed lineage leukemia 1 (MLL1)–WDR5 interaction

WDR5 is an essential protein for enzymatic activity of MLL1. Targeting the protein–protein interaction (PPI) between MLL1 and WDR5 represents a new potential therapeutic strategy for MLL leukemia. Based on the structure of reported inhibitor WDR5-0103, a class of ester compounds were designed and synthetized to disturb MLL1–WDR5 PPI. These inhibitors efficiently inhibited the histone methyltransferase activity in vitro. Especially, WL-15 was one of the most potent inhibitors, blocking the interaction of MLL1–WDR5 with IC₅₀ value of 26.4 nM in competitive binding assay and inhibiting the catalytic activity of MLL1 complex with IC₅₀ value of 5.4 μM. Docking model indicated that ester compounds suitably occupied the central cavity of WDR5 protein and recapitulated the interactions of WDR5-0103 and the hydrophobic groups and key amino greatly increased the activity in blocking MLL1–WDR5 PPI.     

Advances in protein–amino acid nutrition of poultry

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

Probing linker design in citric acid–ciprofloxacin conjugates

A series of structurally related citric acid–ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.     

Molecular interference in antibody–antigen interaction studied with magnetic force immunoassay

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Effect of electrolytes and of distilled water on antigen–antibody complexes

Specific immune precipitates dissolve in concentrated solutions of alkali-metal halides, and of alkaline-earth-metal halides and thiocyanates. The quantity of protein dissolved depends on the nature of the antigen-antibody system, on the proportion of the antigen in the precipitate, and on the avidity of the antibody. The extent of solubilization is a function of the temperature, of the volume of solution used and of the concentration of the ions in the solution, and also depends on the nature of these ions. The dissolving power of bivalent cations is greater than that of monovalent ones, and is as follows: Mg(2+)[unk]Ba(2+)[unk]Ca(2+)[unk]Sr(2+). Antigen-antibody complexes and free antibodies, but no free antigen, are detected in supernatants of specific precipitates dissolved in solutions of electrolytes of low ionic strength. Antigen-antibody complexes, free antibodies and also free antigen are detected in supernatants of specific precipitates dissolved in solutions of electrolytes of high ionic strength. Comparable results are obtained when the electrolyte solutions are studied for their effect on the bonds formed between an antibody and its corresponding immunosorbent. Moreover, in the latter case, 50% of the fixed antibodies could be recovered by elution with distilled water.     

Distinct amino acid–sensing mTOR pathways regulate skeletal myogenesis

Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase–independent manner. Our results uncover amino acid–sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.     

Synthesis and antibacterial evaluation of amino acid–antibiotic conjugates

Amino acid conjugates of quinolone, metronidazole and sulfadiazine antibiotics were synthesized in good yields using benzotriazole methodology. All the conjugates were screened for their antibacterial activity using methods adapted from the Clinical and Laboratory Standards Institute. Antibiotic conjugates were tested for activity in four medically relevant organisms; Staphylococcus aureus (RN4220), Escherichia coli (DH5α), Pseudomonas aeruginosa (PAO1), and Bacillus subtilis (168). Several antibiotic conjugates show promising results against several of the strains screened.