Cross-Reactive Synbody Affinity Ligands for Capturing Diverse Noroviruses.

Noroviruses are the most common cause of acute gastroenteritis in the developed world. Noroviruses are a diverse group of nonenveloped RNA viruses that are continuously evolving. This leads to the rise of immunologically distinct strains of the same genotype on a frequent basis. This diversity presents a unique challenge for detection and tracking of new strains, with the continuous need for new norovirus affinity ligands. Our group developed a family of bivalent synbody affinity ligands using a virus-like particle (VLP) from the 2006 GII.4 Minerva strain of norovirus. We produced more than 20 synbodies with low nanomolar dissociation constants (KD < 10 nM) for GII.4 VLP. We measured binding affinity for four synbodies against VLPs from multiple GI and GII genotypes and found that the synbodies were broadly cross-reactive with affinities that ranged from 0.5 to 8 nM. We tested the ability of these synbodies to capture norovirus from dilute solutions and found that one synbody could capture GII.4 from a 200 000-fold dilution from a norovirus positive stool sample. When these synbodies were tested for the ability to capture of multiple genotypes, we found that four different genotypes were recognized. These data demonstrate that the synbody approach can generate multiple affinity ligands for future use in norovirus detection and possible therapeutic development.

Antineoplastic agents. 599. Total synthesis of dolastatin 16.

The first 23-step total synthesis of the cyclodepsipeptide dolastatin 16 (1) has been achieved. Synthesis of the dolaphenvaline and dolamethylleuine amino acid units using simplified methods improved the overall efficiency. The formation of the 25-membered macrocycle employing lactonization with 2-methyl-6-nitrobenzoic anhydride completed a key step in the synthesis. Regrettably, the synthetic dolastatin 16 (1), while otherwise identical (by X-ray crystal structure and spectral analyses) with the natural product, did not reproduce the powerful (nanomolar) cancer cell growth inhibition displayed by the natural isolate. Presumably this result can be attributed to conformation(s) of the synthetic dolastatin 16 (1) or to a chemically undetected component isolated with the natural product.

Medical migration within Europe: opportunities and challenges.

The free movement of European citizens to live and work within the European Union (EU) is one of the fundamental pillars of the European single market. Recent EU legislation on the recognition of professional qualifications (to take effect January 2016) updates the framework within which doctors and others can migrate freely between EU member states to practise their profession. UK organisations lobbied extensively to change aspects of the original proposals, in particular those that threatened to 'water down' public protection in the interest of free movement. The legislation finally adopted significantly increases safeguards for patients and the public. The revised law covers the rules to be applied by regulators on (for example) assuring language competence, warning 'blacklists' of practitioners subject to sanctions, 'fast track' registration based on mutual recognition of professional qualifications, agreed minimum education and training requirements for mutual recognition, and encouragement of continuing professional development. Drafting of detailed secondary legislation is ongoing and poses opportunities and challenges for patient safety, quality of care and transparency.

Austerity: a failed experiment on the people of Europe.

Many governments in Europe, either of their own volition or at the behest of the international financial institutions, have adopted stringent austerity policies in response to the financial crisis. By contrast, the USA launched a financial stimulus. The results of these experiments are now clear: the American economy is growing and those European countries adopting austerity, including the UK, Ireland, Greece, Portugal and Spain, are stagnating and struggling to repay rising debts. An initial recovery in the UK was halted once austerity measures hit. However, austerity has been not only an economic failure, but also a health failure, with increasing numbers of suicides and, where cuts in health budgets are being imposed, increasing numbers of people being unable to access care. Yet their stories remain largely untold. Here, we argue that there is an alternative to austerity, but that ideology is triumphing over evidence. Our paper was written to contribute to discussions among health policy leaders in Europe that will take place at the 15th European Health Forum at Gastein in October 2012, as its theme 'Crisis and Opportunity - Health in an Age of Austerity'.

Engineering a synthetic ligand for tumor necrosis factor-alpha.

One approach to prepare protein binding ligands is to join two low-affinity ligands that bind different sites on the target protein to create a high-affinity bivalent ligand. This typically requires some knowledge of the ligand binding site and requires exquisite orientation of the ligands in order to achieve maximum binding affinity. Here, we explored the limit of affinity improvement possible with no a priori knowledge of peptide binding site and with minimal effort spent in linking the lead peptides. We compared the affinity enhancement from linking two peptides with low affinity for tumor necrosis factor-α (TNFA) to the affinity enhancement from linking affinity improved versions of these peptides using several different scaffolds. We found that we achieved the highest affinity gain not by the precise positioning of the peptides, but rather by using affinity improved versions of the lead peptides to produce synbodies with apparent K(D)'s of 9 to 48 nM. Kinetic analysis showed that the binding kinetics of the synbody are strongly influenced by the kinetics of the starting peptide. This suggests that careful selection of peptides based on their kinetic profile prior to linking will influence the kinetics of the final binding agent.

Antineoplastic agents. 590. X-ray crystal structure of dolastatin 16 and syntheses of the dolamethylleuine and dolaphenvaline units.

Three advances necessary to bring dolastatin 16 (1) into full-scale preclinical development as an anticancer drug have been accomplished. The X-ray crystal structure of dolastatin 16 has been solved, which allowed stereoselective syntheses of its two new amino acid units, dolamethylleuine (Dml) and dolaphenvaline (Dpv), to be completed. The X-ray crystal structures of synthetic Z-Dml and TFA-Dpv have also been completed.

Thermodynamic additivity of sequence variations: an algorithm for creating high affinity peptides without large libraries or structural information.

BACKGROUND: There is a significant need for affinity reagents with high target affinity/specificity that can be developed rapidly and inexpensively. Existing affinity reagent development approaches, including protein mutagenesis, directed evolution, and fragment-based design utilize large libraries and/or require structural information thereby adding time and expense. Until now, no systematic approach to affinity reagent development existed that could produce nanomolar affinity from small chemically synthesized peptide libraries without the aid of structural information. METHODOLOGY/PRINCIPAL FINDINGS: Based on the principle of additivity, we have developed an algorithm for generating high affinity peptide ligands. In this algorithm, point-variations in a lead sequence are screened and combined in a systematic manner to achieve additive binding energies. To demonstrate this approach, low-affinity lead peptides for multiple protein targets were identified from sparse random sequence space and optimized to high affinity in just two chemical steps. In one example, a TNF-α binding peptide with K(d) = 90 nM and high target specificity was generated. The changes in binding energy associated with each variation were generally additive upon combining variations, validating the basis of the algorithm. Interestingly, cooperativity between point-variations was not observed, and in a few specific cases, combinations were less than energetically additive. CONCLUSIONS/SIGNIFICANCE: By using this additivity algorithm, peptide ligands with high affinity for protein targets were generated. With this algorithm, one of the highest affinity TNF-α binding peptides reported to date was produced. Most importantly, high affinity was achieved from small, chemically-synthesized libraries without the need for structural information at any time during the process. This is significantly different than protein mutagenesis, directed evolution, or fragment-based design approaches, which rely on large libraries and/or structural guidance. With this algorithm, high affinity/specificity peptide ligands can be developed rapidly, inexpensively, and in an entirely chemical manner.

Discovery of high-affinity protein binding ligands--backwards.

BACKGROUND: There is a pressing need for high-affinity protein binding ligands for all proteins in the human and other proteomes. Numerous groups are working to develop protein binding ligands but most approaches develop ligands using the same strategy in which a large library of structured ligands is screened against a protein target to identify a high-affinity ligand for the target. While this methodology generates high-affinity ligands for the target, it is generally an iterative process that can be difficult to adapt for the generation of ligands for large numbers of proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a class of peptide-based protein ligands, called synbodies, which allow this process to be run backwards--i.e. make a synbody and then screen it against a library of proteins to discover the target. By screening a synbody against an array of 8,000 human proteins, we can identify which protein in the library binds the synbody with high affinity. We used this method to develop a high-affinity synbody that specifically binds AKT1 with a K(d)<5 nM. It was found that the peptides that compose the synbody bind AKT1 with low micromolar affinity, implying that the affinity and specificity is a product of the bivalent interaction of the synbody with AKT1. We developed a synbody for another protein, ABL1 using the same method. CONCLUSIONS/SIGNIFICANCE: This method delivered a high-affinity ligand for a target protein in a single discovery step. This is in contrast to other techniques that require subsequent rounds of mutational improvement to yield nanomolar ligands. As this technique is easily scalable, we believe that it could be possible to develop ligands to all the proteins in any proteome using this approach.

High-throughput screening in two dimensions: binding intensity and off-rate on a peptide microarray.

We report a high-throughput two-dimensional microarray-based screen, incorporating both target binding intensity and off-rate, which can be used to analyze thousands of compounds in a single binding assay. Relative binding intensities and time-resolved dissociation are measured for labeled tumor necrosis factor alpha (TNF-alpha) bound to a peptide microarray. The time-resolved dissociation is fitted to a one-component exponential decay model, from which relative dissociation rates are determined for all peptides with binding intensities above background. We show that most peptides with the slowest off-rates on the microarray also have the slowest off-rates when measured by surface plasmon resonance (SPR).

Creating protein affinity reagents by combining peptide ligands on synthetic DNA scaffolds.

A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps. Central to this process is the idea that small peptide libraries contain sequences that will bind to independent regions on a protein surface and that these ligands can be combined on synthetic scaffolds to create high affinity bivalent reagents. To demonstrate the feasibility of this approach, an array of 4000 unique 12-mer peptides was screened to identify sequences that bind to nonoverlapping sites on the yeast regulatory protein Gal80. Individual peptide ligands were screened at different distances using a novel DNA linking strategy to identify the optimal peptide pair and peptide pair separation distance required to transform two weaker ligands into a single high affinity protein capture reagent. A synthetic antibody or synbody was created with 5 nM affinity to Gal80 that functions in conventional ELISA and pull-down assays. We validated our synthetic antibody approach by creating a second synbody to human transferrin. In both cases, we observed an increase in binding affinity of approximately 1000-fold (DeltaDeltaG = approximately 4.1 kcal/mol) between the individual peptides and final bivalent synbody construct.