Michal Lebl

Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens.

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR↓GL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1′ were more selective for furin. Peptides with P1′ Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1-P2-P3-P4-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1 or the P2 site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

A multiplexed protein kinase assay.

We report a novel protein kinase assay designed for high‐throughput detection of one or many kinases in a complex mixture. A solution‐phase phosphorylation reaction is performed on 900 different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation, phosphoserine, phosphothreonine, and phosphotyrosine are chemically labeled, and the substrates are hybridized to a microarray with oligonucleotides complementary to the tags to read out the phosphorylation state of each peptide. Because protein kinases act on more than one peptide sequence, each kinase can be characterized by a unique signature of phosphorylation activity on multiple substrates. Using this method, we determined signatures for 26 purified kinases and demonstrated that enzyme mixtures can be screened for activity and selectivity of inhibition.

High-Throughput Peptide Synthesis

The methodologies of high-throughput peptide synthesis are overviewed and discussed. Particular focus is given to the techniques applicable to laboratories with a limited budget. Automated solutions for synthetic problems are also discussed.

Fully Automated Parallel Oligonucleotide Synthesizer

The oligonucleotide and peptide synthesis technology is of major strategic importance in the field of genomics and proteomics. Commercially available single channel synthesizers cannot satisfy the demand of emerging technologies. Currently, there are several instruments for parallel synthesis of oligonucleotides such as: (i) a 96-channel instrument based on a microtiter plate format developed by scientists at Stanford University [1]; (ii) PolyPlex machine produced by the company GeneMachines [2]; or synthesizer using two microtiterplates for simultaneous synthesis of 192 oligonucleotides, developed at The University of Texas Southwestern Medical Center at Dallas, and sold under name MerMade by company BioAutomation [3]. While these technologies meet the modest requirements of most experiments today, they are inadequate for the manufacturing needs looming in the very near future. Current synthesis technologies do not meet the need for manufacturing large numbers of oligonucleotides (tens of thousands to millions of sequences) cost-effectively. Our goal was to fill this gap and build the parallel (and economical) synthesizer capable of preparation of needed numbers of oligonucleotides.

Evaluation of different chemical strategies for conjugation of oligonucleotides to peptides.

We developed novel assays for high-throughput detection of one or many kinases or proteases. The assays use hundreds of different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation with sample, the pool of substrates is hybridized to a microarray containing oligonucleotides complementary to the tag sequences. We screened several specific chemistries for the conjugation based on the following criteria: easy derivatization of oligonucleotides and peptides; high efficiency of the conjugation reaction; good stability of the conjugates; and satisfactory conjugate performance in our assays. We have validated selected method during the successful generation of thousands oligonucleotide-peptide conjugates.

Economical Parallel Oligonucleotide and Peptide Synthesizer – Pet Oligator

We have developed a small benchtop oligonucleotide synthesizer which allows the scientist to prepare, rapidly and economically, up to 24 oligonucleotides in one batch. We have shown that this instrument can be used for peptide synthesis, as well. The instrument is based on the centrifugation method for solid–liquid separation.

Neuromorphic Processing for Optical Microbead Arrays: Dimensionality Reduction and Contrast Enhancement

This paper presents a neuromorphic approach for sensor-based machine olfaction that combines a portable chemical detection system based on microbead array technology with a biologically inspired model of signal processing in the olfactory bulb. The sensor array contains hundreds of microbeads coated with solvatochromic dyes adsorbed in, or covalently attached on, the matrix of various microspheres. When exposed to odors, each bead sensor responds with corresponding intensity changes, spectral shifts, and time-dependent variations associated with the fluorescent sensors. The bead array responses are subsequently processed using a model of olfactory circuits that capture the following two functions: chemotopic convergence of receptor neurons and center on-off surround lateral interactions. The first circuit performs dimensionality reduction, transforming the high-dimensional microbead array response into an organized spatial pattern (i.e., an odor image). The second circuit enhances the contrast of these spatial patterns, improving the separability of odors. The model is validated on an experimental dataset containing the responses of a large array of microbead sensors to five different analytes. Our results indicate that the model is able to significantly improve the separability between odor patterns, compared to that available from the raw sensor response

Synthesis of poly d(G-C) oligonucleotides.

Model sequences for evaluation of the GC dimer sequence repetition on synthesis success were prepared and analyzed by HPLC. Contiguous d(G-C) or d(C-G) sequences have a deleterious effect on DNA oligonucleotide synthesis. The critical number seems to be about 6 GCs in a row. If the GCs are separated by other nucleotides, the effect is not as severe.