The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.

Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo . Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ ² can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb ³⁺ - TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions—fluorescence lifetime up to milliseconds—to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo .

Insulin receptor–related receptor (IRR) is a receptor tyrosine kinase of the insulin receptor family and functions as an extracellular alkali sensor that controls metabolic alkalosis in the regulation of the acid–base balance. In the present work, we sought to analyze structural features of IRR by comparing them with those of the insulin receptor, which is its closest homolog but does not respond to pH changes. Using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), we investigated the overall conformation of the recombinant soluble IRR ectodomain (ectoIRR) at neutral and alkaline pH. In contrast to the well-known inverted U-shaped (or λ-shaped) conformation of the insulin receptor, the structural models reconstructed at different pH values revealed that the ectoIRR organization has a “droplike” shape with a shorter distance between the fibronectin domains of the disulfide-linked dimer subunits within ectoIRR. We detected no large-scale pH-dependent conformational changes of ectoIRR in both SAXS and AFM experiments, an observation that agreed well with previous biochemical and functional analyses of IRR. Our findings indicate that ectoIRR's sensing of alkaline conditions involves additional molecular mechanisms, for example engagement of receptor juxtamembrane regions or the surrounding lipid environment.

Spirocyclic compounds containing heterocyclic moieties represent promising 3D scaffolds for modern drug design. In the search for novel anti-flaviviral agents, we have obtained a series of 3-[N,N-bis(sulfonyl)amino]isoxazolines containing spiro-annulated cyclooctane rings and assessed their antiviral activity against tick-borne encephalitis (TBEV), yellow fever (YFV), and West Nile (WNV) viruses. The structural analogs of spirocyclic compounds with a single sulfonyl group or 1,2-annulated cyclooctane ring were also investigated. Almost all the studied 3-[N,N-bis(sulfonyl)amino]isoxazolines revealed antiviral activity against TBEV and WNV. The most active against TBEV was spiro-isoxazoline derivative containing p-nitrophenyl groups in the sulfonyl part (EC₅₀ 2.0 ± 0.5 μM), while the highest potency against WNV was found for the compounds with lipophilic substituents in sulfonyl moiety, naphtyl being the most favorable one (EC₅₀ 1.3 ± 0.5 μM). In summary, two novel scaffolds of anti-flaviviral agents based on N,N-bis(sulfonyl)amino]isoxazoline were proposed, and the compounds of this type demonstrated activity against TBEV and WNV.