CD73 is a crucial regulator of adenosine production in the tumor microenvironment and, therefore, represents a valuable target for cancer immunotherapy. While different inhibitors of CD73 have been studied, the progress remains hindered by a lack of high-throughput assays that would allow the screening of large chemical libraries. Establishing a sensitive assay for the detection of CD73 activity could enable additions to the CD73 inhibitor chemical space as well as help facilitate a better understanding of the CD73 reaction mechanism. In this study, we focused on the development and adaptation of DIANA for CD73 high-throughput screening and showed that we can detect enzyme inhibition with high sensitivity. We then used this assay to screen an IOCB library, a proprietary set of chemical compounds with a special focus on nucleotide analogues. We identified several scaffolds that inhibit CD73 and in an SAR study demonstrated fine-tuning of the inhibition properties of monophosphonate analogues. Moreover, using a breast cancer cell line as a model with endogenous CD73 expression, we demonstrated the inhibition of CD73 directly on cells. The establishment of a sensitive assay for the detection of CD73 activity allowed us to develop potent inhibitors of the enzyme with low nanomolar inhibition constants. Our findings further promote the importance of CD73 inhibitors in cancer therapy.

• Keywords
• CD73, DNA-linked probe, ecto-5′-nucleotidase, high-throughput screening, monophosphonate, tumor microenvironment,
• Publication type
• Journal Article MeSH

Given its highly innovative character and potential socioeconomic impact, Synthetic Biology is often ranked among prominent research areas and national research priorities in developed countries. The global evolution of this field is proceeding by leaps and bounds but its development at the level of individual states varies widely. Despite their current satisfactory economic status, the majority of 13, mostly post-communist, countries that entered the European Union family in and after 2004 (EU13) have long overlooked the blossoming of Synthetic Biology. Their prioritized lines of research have been directed elsewhere or "Synthetic Biology" did not become a widely accepted term to encompass their bioengineering and biotechnology domains. The Czech Republic is not an exception. The local SynBio mycelium already exists but is mainly built bottom-up through the activities of several academic labs, iGEM teams, and spin-off companies. In this article, we tell their individual stories and summarize the prerequisites that allowed their emergence in the Czech academic and business environment. In addition, we provide the reader with a brief overview of laboratories, research hubs, and companies that perform biotechnology and bioengineering-oriented research and that may be included in a notional "shadow SynBio community" but have not yet adopted Synthetic Biology as a unifying term for their ventures. We also map the current hindrances for a broader expansion of Synthetic Biology in the Czech Republic and suggest possible steps that should lead to the maturity of this fascinating research field in our country.

• Keywords
• Biotechnology and bioengineering, Community, Czech Republic, EU13 countries, Public perception, Research landscape, Synthetic biology, iGEM,
• Publication type
• Journal Article MeSH

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

• Keywords
• DIANA, assay, crystallography, influenza neuraminidase,
• MeSH
• Antiviral Agents chemistry   pharmacology   MeSH
• Influenza, Human drug therapy   enzymology   virology   MeSH
• DNA chemistry   MeSH
• Enzyme Inhibitors chemistry   pharmacology   MeSH
• Phosphorous Acids chemistry   MeSH
• Humans MeSH
• Neuraminidase antagonists & inhibitors   metabolism   MeSH
• Oseltamivir analogs & derivatives   chemistry   MeSH
• Drug Evaluation, Preclinical methods   MeSH
• Reproducibility of Results MeSH
• Viral Proteins antagonists & inhibitors   metabolism   MeSH
• Influenza A virus drug effects   enzymology   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antiviral Agents MeSH
• DNA MeSH
• Enzyme Inhibitors MeSH
• Phosphorous Acids MeSH
• Neuraminidase MeSH
• Oseltamivir MeSH
• tamiphosphor MeSH Browser
• Viral Proteins MeSH