Fragment-based discovery of novel thymidylate synthase leads by NMR screening and group epitope mapping

Solution-state nuclear magnetic resonance (NMR) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the cofactor-binding pocket of hTS. Screening hits were used as search criteria within commercially available sources, and a subset of catalog analogs were tested for potency by in vitro assay and binding affinity by quantitative saturation transfer difference (STD)-NMR titration. Two compounds identified by this approach possess low micromolar affinity and potency, as well as excellent binding efficiency against hTS. Relative binding orientations for both leads were modeled using AutoDock, and the most likely bound conformations were validated using experimentally derived STD-NMR binding epitope data. These ligands represent novel starting points for fragment-based drug design of non-canonical TS inhibitors, and their binding epitopes highlight important and previously unexploited interactions with conserved residues in the cofactor-binding site.     

Structural and immunogenicity analysis of chimeric B‐cell epitope constructs derived from the gp46 and gp21 subunits of the envelope glycoproteins of HTLV‐1

Abstract: B‐cell epitopes were selected from the gp21 and gp46 subunits of the envelope glycoprotein of human T‐cell lymphotropic virus type 1 (HTLV‐1) by computer‐aided analyses of protein antigenicity. Molecular modeling was used to design and synthesize the epitopes as chimeric constructs with promiscuous T‐helper epitopes derived either from the tetanus toxoid (amino acids 947–967) or measles virus fusion protein (amino acids 288–302). Circular dichroism measurements revealed that the peptides had a secondary structure that correlated well with the crystal structure data or predicted structure. The chimeric peptides were then evaluated for their immunogenicity in rabbits or mice. Antibodies against one of the epitopes derived from the gp21 subunit were found to be neutralizing in its ability to inhibit the formation of virus‐induced syncytia. These studies underscore the importance of the gp21 transmembrane region for the development of vaccine candidates. The applicability of a chimeric approach is discussed in the context of recent findings regarding the role of gp21 transmembrane region in the viral fusion process.     

Evolution of phage display technology: from discovery to application

Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.     

Analogue synthesis reveals decoupling of antibiofilm and β‐lactam potentiation activities of a lead 2‐aminoimidazole adjuvant against Mycobacterium smegmatis

Biofilm formation is one of the many mechanisms bacteria utilize to survive antibiotic treatment. It has been demonstrated that when Mycobacterium tuberculosis exists in a biofilm in vitro, it expresses phenotypic resistance to antimicrobial drugs. As the in vivo survival of M. tuberculosis following drug treatment is potentially linked to a biofilm‐like expression of drug tolerance, it is hypothesized that biofilm dispersion should increase antibiotic susceptibility and reduce the duration of the current antibiotic treatment regimen. Previously, we have identified a 2‐aminoimidazole (2‐AI) compound capable of dispersing and inhibiting M. tuberculosis and M. smegmatis biofilms in vitro. Additionally, this compound potentiated the activity of carbenicillin against M. tuberculosis and, to a lesser degree, M. smegmatis. Here, we describe a SAR study on this compound evaluating each derivative for biofilm dispersion and β‐lactam potentiation capabilities against M. smegmatis. This study identified a compound that improved upon the biofilm dispersion capabilities of the lead compound. Interestingly, a different compound was identified with an increased ability to potentiate a subset of β‐lactam antibiotics. These compounds indicate that biofilm dispersion and potentiation capabilities may not be associated.     

Comparative epitope mapping with saturation transfer difference NMR of sialyl Lewis(a) compounds and derivatives bound to a monoclonal antibody

The monoclonal antibody GSLA-2 recognizes the sialyl Lewis(a) (sLe(a)) epitope, which has an increased occurrence on mucin type glycoproteins of patients with colorectal carcinoma. GSLA-2 is therefore used in tumor diagnosis. To advance the understanding of this highly specific molecular recognition reaction, we have analyzed the binding epitope of sLe(a) at atomic resolution using saturation transfer difference NMR. To compare, the binding epitopes of sialyl Lewis(x) (sLe(x)) and of four synthetic derivatives of sLe(a) were explored. Surface plasmon resonance experiments furnished thermodynamic and kinetic data. It is observed that all pyranose rings of sLe(a) are in contact with the protein surface, with the neuramic acid residue receiving the largest fraction of saturation transfer. In contrast, sLe(x) binds very weakly, though specifically to GSLA-2, with a different binding epitope. This study provides a comprehensive picture of the recognition sLe(a) and explains the exquisite specificity of the antibody.     

Structure−affinity relationships in the gp41 ELDKWA epitope for the HIV‐1 neutralizing monoclonal antibody 2F5: effects of side‐chain and backbone modifications and conformational constraints

Abstract: The human monoclonal antibody, mAb 2F5, has broad HIV‐1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high‐affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme‐linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end‐capped, linear nonapeptide, Ac‐LELDKWASL‐amide, was sufficient to attain maximal affinity within the set of native gp41‐sequence peptides assayed. Scanning single‐residue alanine and d ‐residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high‐affinity binding of the linear nonapeptide. Further studies of side‐chain and backbone‐modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu³ and Trp⁶ residues. The nine‐residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15‐residue, disulfide‐bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C‐peptide helix immediately adjacent to the N‐terminal end of the ELDKWA epitope were examined through the synthesis of helix‐initiated analogs. Circular dichroism (CD) studies indicated that the α‐helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV‐neutralizing actions of 2F5.     

Characterization of the interaction forces in a drug carrier complex of doxorubicin with a drug‐binding peptide

Polypeptide‐based materials are used as building blocks for drug delivery systems aimed at toxicity decrease in chemotherapeutics. A molecular‐level approach is adopted for investigating the non‐covalent interactions between doxorubicin and a recently synthesized drug‐binging peptide as a key part of a system for delivery to neoplastic cells. Molecular dynamics simulations in aqueous solution at room and body temperature are applied to investigate the structure and the binding modes within the drug–peptide complex. The tryptophans are outlined as the main chemotherapeutic adsorption sites, and the importance of their placement in the peptide sequence is highlighted. The drug–peptide binging energy is evaluated by density functional theory calculations. Principal component analysis reveals comparable importance of several types of interaction for the binding strength. π‐Stacking is dominant, but other factors are also significant: intercalation, peptide backbone stacking, electrostatics, dispersion, and solvation. Intra‐ and intermolecular H‐bonding also stabilizes the complexes. The influence of solvent molecules on the binding energy is mild. The obtained data characterize the drug‐to‐peptide attachment as a mainly attractive collective process with interactions spanning a broad range of values. These results explain with atomistic detail the experimentally registered doxorubicin‐binging ability of the peptide and outline the complex as a prospective carrying unit that can be employed in design of drug delivery systems.     

Validation of a sample pretreatment protocol to convert a drug‐sensitive into a drug‐tolerant anti‐infliximab antibody immunoassay

A meta‐analysis revealed that up to 51% of patients treated with infliximab develop anti‐drug Abs (ADA) which are associated with loss of response. Detection of ADA is strongly influenced by assay technique since drug‐sensitive ADA assays are not able to detect ADA in the presence of drug and therefore underestimate ADA development. In addition, the lack of a calibrator antibody that can be used in a drug‐sensitive and drug‐tolerant assay hampers an adequate comparison among different assays. Here we present a sample pretreatment protocol to convert the bridging assay, originally developed as a drug‐sensitive assay, into a drug‐tolerant assay, allowing use of the same assay and calibrator antibody MA‐IFX10F9. Using the sample pretreatment protocol, the bridging assay detects antibodies towards infliximab in samples containing up to 5‐fold infliximab over anti‐infliximab. Analysis of consecutive serum samples from infliximab treated patients revealed that the drug‐tolerant assay detects ADA development up to 40 weeks earlier compared to the drug‐sensitive assay. In conclusion, the sample pretreatment protocol can be implemented in various assay formats and allows determination of ADA in the presence of drug, providing the possibility for early treatment optimization. Copyright © 2016 John Wiley & Sons, Ltd.     

Four phase 1 trials to evaluate the safety and pharmacokinetic profile of single and repeated dosing of SCO‐101 in adult male and female volunteers

SCO‐101 (Endovion) was discontinued 20 years ago as a new drug under development against sickle cell anaemia. Data from the phase 1 studies remained unpublished. New data indicate that SCO‐101 might be efficacious as add‐on therapy in cancer. Thus, we report the results from the four phase 1 trials performed between 2001 and 2002. Adult volunteers received SCO‐101 or placebo in four independent trials. Adverse events were recorded, and SCO‐101 was determined for pharmacokinetic analysis. Ninety‐two volunteers completed the trials. The most remarkable adverse effect was a transient and dose‐dependent increase in unconjugated bilirubin. Plasma SCO‐101 elimination was approximately log linear, with apparent oral clearances of between 315 and 2103 mL/h for single doses, and between 121 and 2433 mL/h at steady state following oral administration. There was a marked decrease in clearance with increasing dose, and for repeated dose versus single dose. T_max was greater, and C_max and AUC_∞ were lower in the fed state compared to the fasted state. Exposure was equivalent in males and females and for African Americans and Caucasians. In conclusion, SCO‐101 appears to be a safe drug with a predictable PK profile. Its efficacy as add‐on to standard anticancer drugs has yet to be defined.     

Searching for Dual Inhibitors of the MDM2‐p53 and MDMX‐p53 Protein–Protein Interaction by a Scaffold‐Hopping Approach

Two libraries of substituted benzimidazoles were designed using a ‘scaffold‐hopping’ approach based on reported MDM2‐p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X‐ray structure. Benzimidazole libraries were prepared using an efficient solution‐phase approach and screened for inhibition of the MDM2‐p53 and MDMX‐p53 protein–protein interactions. Key examples showed inhibitory activity against both targets.     

Virulence‐targeted Antibacterials: Concept,Promise, and Susceptibility to Resistance Mechanisms

In view of the relentless increase in antibiotic resistance in human pathogens, efforts are needed to safeguard our future therapeutic options against infectious diseases. In addition to regulatory changes in our antibiotic use, this will have to include the development of new therapeutic compounds. One area that has received growing attention in recent years is the possibility to treat or prevent infections by targeting the virulence mechanisms that render bacteria pathogenic. Antivirulence targets include bacterial adherence, secretion of toxic effector molecules, bacterial persistence through biofilm formation, quorum sensing and immune evasion. Effective small‐molecule compounds have already been identified that suppress such processes. In this review, we discuss the susceptibility of such compounds to the development of resistance, by comparison with known resistance mechanisms observed for classical bacteriostatic or bacteriolytic antibiotics, and by review of available experimental case studies. Unfortunately, appearance of resistance mechanisms has already been demonstrated for some, showing that the quest of new, lasting drugs remains complicated.     

Novel Glycoconjugate of 8‐Fluoro Norfloxacin Derivatives as Gentamicin‐resistant Staphylococcus aureus Inhibitors: Synthesis and Molecular Modelling Studies

Antibiotic resistance has been the subject of interest in clinical practice due to high prevalence of antibiotic‐resistant pathogenic organisms. In view of the prevalence of lesser resistance in antibiotics belonging to aminoglycoside class of compounds viz. Food and Drug Administration‐approved gentamicin for the treatment of Staphylococcus infections, which also has instances of resistance in the clinical isolates of Staphylococcus aureus, a series of novel glycoconjugates of 8‐fluoro norfloxacin analogues with high regio‐selectivity by employing copper (I)‐catalyzed 1, 3‐dipolar cycloaddition of 1‐O‐propargyl monosaccharides has been synthesized and evaluated for the antibacterial activity against gentamicin resistance Staphylococcus aureus. Among these compounds, the compound 10g showed better antibacterial activity (MIC = 3.12  μ g/ml) than gentamicin (Escherichia coli (12.5  μ g/ml), Staphylococcus aureus (6.25  μ g/ml) and Klebsiella pneumonia (6.25  μ g/ml), including gentamicin resistant (>50  μ g/ml) strain in vitro). The docking studies suggest DNA gyrase of Staphylococcus aureus as a probable target for the antibacterial action of compound 10g .     

siRNA Delivery: From Lipids to Cell‐penetrating Peptides and Their Mimics

To deliver siRNA for therapeutic use, several hurdles must be addressed. Metabolic degradation must be blocked, and the RNAi cellular machinery is located in the cytoplasm, while double‐stranded siRNA is large, highly charged and impermeable to cell membranes. To date, the solutions to the delivery issues have mostly involved different forms of lipid particle encapsulation. Cell‐penetrating peptides and their mimics or analogues offer a different approach and this is an emerging field with the first in vivo examples now reported. Recent reports point to lipid receptors being involved in the cellular uptake of both types of transporter. This review examines the delivery of siRNA with a focus on cell‐penetrating peptides and their small molecule and oligomeric mimics. The current status of siRNA delivery methods in clinical trials is examined. It now seems that the goal of delivering siRNA therapeutically is achievable but will they form part of a sustainable healthcare portfolio for the future.     

From pro defensins to defensins: synthesis and characterization of human neutrophil pro α‐defensin‐1 and its mature domain

Abstract: Human neutrophil α‐defensins (HNPs) are small, cationic, Cys‐rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre‐proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro‐peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45‐residue pro‐peptide and the C‐terminal functional domain. Here we described, total chemical synthesis of the 75‐residue human neutrophil pro α‐defensin‐1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met⁴⁵–Ala⁴⁶ peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys¹–Cys⁶, Cys²–Cys⁴ and Cys³–Cys⁵, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro‐peptide binds to HNP1 intermolecularly with an apparent K_d value of 6.2 μm at pH 7.4, confirming the mode of intramolecular inactivation of human α‐defensin precursors.     

Synthesis and immunological studies of α‐conotoxin chimera containing an immunodominant epitope from the 268–284 region of HSV gD protein

Abstract: We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as ‘guest’ sequence in the ‘host’ structure of α‐conotoxin GI, a 13‐residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276–284 region of HSV gD‐1 selected for these studies is highly hydrophilic and adopts a β‐turn. The α‐conotoxin GI also contains a β‐turn in the 8–12 region, stabilized by two disulfide bridges at positions 2–7 and 3–13. Thus, the tetramer sequence of α‐conotoxin, ⁸Arg‐His‐Tyr‐Ser¹² has been replaced by Asp‐Pro‐Val‐Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3–13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys²‐Cys⁷ three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 m HCl or Tl(tfa)₃ in trifluoroacetic acid (TFE)] were compared. The high‐performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimeric peptide and native α‐conotoxin GI showed similar circular dichroism spectra in phosphate‐buffered saline (PBS) and in a PBS‐TFE 1 : 1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M‐ and IgG‐type antibody responses showed that the bicyclic HSV‐α‐[Tyr¹]‐conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV‐α‐[Tyr¹]‐conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV‐α‐[Tyr¹]‐conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG‐specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.     

Induction of carbohydrate‐specific antibodies in HLA‐DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use

Abstract: Over the last few years, anticancer immunotherapy has emerged as a new exciting area for controlling tumors. In particular, vaccination using synthetic tumor‐associated antigens (TAA), such as carbohydrate antigens hold promise for generating a specific antitumor response by targeting the immune system to cancer cells. However, development of synthetic vaccines for human use is hampered by the extreme polymorphism of human leukocyte‐associated antigens (HLA). In order to stimulate a T‐cell dependent anticarbohydrate response, and to bypass the HLA polymorphism of the human population, we designed and synthesized a glycopeptide vaccine containing a cluster of a carbohydrate TAA B‐cell epitope (Tn antigen: α‐GalNAc‐Ser) covalently linked to peptides corresponding to the Pan DR ‘universal’ T‐helper epitope (PADRE) and to a cytotoxic T lymphocyte (CTL) epitope from the carcinoembryonic antigen (CEA). The immunogenicity of the construct was evaluated in outbred mice as well as in HLA transgenic mice (HLA‐DR1, and HLA‐DR4). A strong T‐cell dependent antibody response specific for the Tn antigen was elicited in both outbred and HLA transgenic mice. The antibodies induced by the glycopeptide construct efficiently recognized a human tumor cell line underlying the biological relevance of the response. The rational design and synthesis of the glycopeptide construct presented herein, together with its efficacy to induce antibodies specific for native tumor carbohydrate antigens, demonstrate the potential of a such synthetic molecule as an anticancer vaccine candidate for human use.     

Site‐directed Mutagenesis of Key Residues Unveiled a Novel Allosteric Site on Human Adenosine Kinase for Pyrrolobenzoxa(thia)zepinone Non‐Nucleoside Inhibitors

Most nucleoside kinases, besides the catalytic domain, feature an allosteric domain which modulates their activity. Generally, non‐substrate analogs, interacting with allosteric sites, represent a major opportunity for developing more selective and safer therapeutics. We recently developed a series of non‐nucleoside non‐competitive inhibitors of human adenosine kinase (hAK), based on a pyrrolobenzoxa(thia)zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on hAK, topographically distinct from the catalytic site. In this study, we have adopted a multidisciplinary approach including molecular modeling, biochemical studies, and site‐directed mutagenesis to validate our hypothesis. Based on a three‐dimensional model of interaction between hAK and our molecules, we designed, cloned, and expressed specific, single and double point mutants of hAK (Q74A, Q78A, H107A, K341A, F338A, and Q74A‐F338A). Kinetic characterization of recombinant enzymes indicated that these mutations did not affect enzyme functioning; conversely, mutated enzymes are endowed of reduced susceptibility to our non‐nucleoside inhibitors, while maintaining comparable affinity for nucleoside inhibitors to the wild‐type enzyme. This study represents the first characterization and validation of a novel allosteric site in hAK and may pave the way to the development of novel selective and potent non‐nucleoside inhibitors of hAK endowed with therapeutic potential.     

Role of myelin basic protein epitope MBP74‐85 in experimental autoimmune encephalomyelitis: Elaboration of agonist and antagonist motifs

Conformational properties of the myelin basic protein epitope QKSQRSQDENPV (MBP_74‐85) which can initiate experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis, were investigated by semiempirical methods. Energy calculations were carried out on the full MBP_74‐85 autoantigen and the antagonist analog [Ala⁸¹]MBP_74‐85. These studies have revealed a low energy cyclic conformation for MBP_74‐85 which is characterized by an agonist motif comprising an interaction of the sidechains of Arg⁷⁸ and Asp⁸¹ of MBP_74‐85. Disruption of this agonist motif by removal of the residue 81 carboxylate, as in the antagonist [Ala⁸¹]MBP_74‐85, invokes a compensatory rearrangement of the molecule resulting in interaction of the Arg⁷⁸ sidechain with the sidechain of Glu⁸² together with Lys⁷⁵. This antagonist motif, comprising guanidino, amino, and carboxylate groups, has been reproduced previously in semimimetic peptides having the general structure Ser‐Arg‐LINKER‐Glu‐NH₂ (where LINKER = one or more residues of aminocaproic acid or isonipecotic acid), which have preferred conformations characterized by interaction of the carboxylate with both the guanidino and amino groups in a similar manner to the antagonist motif in [Ala⁸¹]MBP_74‐85. However, in EAE assays these small semimimetics turned out to be partial agonists, i.e., molecules with structures between agonists and antagonists. These findings provide insight into the design of small molecule (orally active) autoantigen antagonists for the treatment of autoimmune diseases such as MS. Drug Dev. Res. 48:1–5, 1999. © 1999 Wiley‐Liss, Inc.