A DNA-encoded macrocyclic peptide library was designed and synthesized with 2.4 × 10 members composed of 4-20 natural and non-natural amino acids. Affinity-based selection was performed against two therapeutic targets, VHL and RSV N protein. On the basis of selection data, some peptides were selected for resynthesis without a DNA tag, and their activity was confirmed.
The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.
A non-radioactive DNA detection chemistry is described and its application is shown for DNA hybridization and standard dideoxy DNA sequencing. The method employes a biotin-streptavidin system which binds an enzyme specifically to a target DNA and upon exposure to substrate, the enzyme catalyzes a chemiluminescent reaction. The image is captured within seconds by a Polaroid or X-ray film. The method is capable of detecting DNA in the hundred attomol range.
Toward a goal of dideoxy sequencing DNA utilizing electrophore labels, we prepared four electrophore-labeled DNA oligonucleotide primers. Each primer has a different electrophore and DNA sequence but a common glycol keto (alpha,beta-dihydroxyketo) release group. Cleavage of this latter group by either periodate oxidation or a thermal retroaldol reaction releases the electrophores for detection by mass spectrometry. Successful sequencing data with these primers was obtained by capillary electrophoresis on an ABI Model 310 after fluorescence dideoxy terminator cycle sequencing reactions were conducted. In a separate experiment, it was demonstrated that a cocktail of the four electrophore DNA primers could be detected as a dried sample spot by CO2 laser desorption/capillary collection/gas chromatography electron capture mass spectrometry. These results establish some feasibility for our long-term goal of high-speed multiplex electrophore mass tag dideoxy DNA sequencing. Ultimately we plan to use a higher number of electrophore mass tags and to rely on direct detection of the desorbed electrophores by electron capture time-of-flight mass spectrometry.
The original version of this Article omitted the following from the Acknowledgements: 'We thank Robert Kirkpatrick for implementing the high throughput protein design strategy that enabled screening and triage of essential A. baumannii targets, based on whole genome sequencing and annotation of BM4454 strain; and Stephanie Van Horn, Allan Kwan, Elizabeth Valoret for A. baumannii genome sequencing and annotation.' Also, the original version omitted an acknowledgement to Prof. Lydia Tabernero as one of our collaborators for supplying the purified proteins used in the Tuberculosis screen. This has been corrected in both the PDF and HTML versions of the Article.
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