Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases

The conjugates of an adenosine mimetic and oligo-l -arginine or oligo-d -arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l -methionine (SAM) and S-adenosyl-l -homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.     

High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK

The bisubstrate fluorescent probe ARC-583 (Adc-Ahx-(d-Arg)₆-d-Lys(5-TAMRA)-NH₂) and its application for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of protein kinases PKA (cyclic AMP-dependent protein kinase) and ROCK (rho kinase) in fluorescence polarization-based assay are described. High affinity of the probe (K_D = 0.48 nM toward PKA) enables its application for the characterization of inhibitors with nanomolar and micromolar potency and determination of the active concentration of the kinase in individual experiments as well as in the high-throughput screening format. The probe can be used for the assessment of protein-protein interactions (e.g., between regulatory and catalytic subunits of PKA) and as a cyclic AMP biosensor.     

Thiazole- and selenazole-comprising high-affinity inhibitors possess bright microsecond-scale photoluminescence in complex with protein kinase CK2

A previously disclosed protein kinase (PK) CK2-selective inhibitor 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) revealed remarkable room temperature phosphorescence when bound to the ATP pocket of the protein kinase CK2. Conjugation of these fragments with a mimic of CK2 substrate peptide resulted in bisubstrate inhibitors with increased affinity towards the kinase. Attachment of the fluorescent acceptor dye 5-TAMRA to the conjugates led to significant enhancement of intensity of long-lifetime (microsecond-scale) photoluminescence of both sulfur- and selenium-containing compounds. The developed photoluminescent probes make possible selective determination of the concentration of CK2 in cell lysates and characterization of CK2 inhibitors by means of time-gated measurement of photoluminescence.     

Day‐flying moths are smaller: evidence for ecological costs of being large

Research on evolutionary forces determining optimal body sizes has primarily relied on experimental evaluation of respective selective pressures. Accounting for among‐species variation through application of phylogenetic comparative methods is a complementary although little used approach. It enables the direct association of body size values with particular environments. Using phylogenetically explicit comparative analyses, we show that small body size is associated with diurnal (rather than nocturnal) activity of adults among temperate species of the moth family Geometridae. The association of an exclusively adult trait with species‐specific body size suggests that optimal body sizes are at least partly determined by the costs being a large adult, as opposed to the more frequently considered costs of attaining large size. It appears likely that size‐selective predation by insectivorous birds is the primary factor responsible for selection against large body size in day‐flying moths.     

Two new Neuratelia Rondani (Diptera,Mycetophilidae) species from Western Palaearctic: a case of limited congruence between morphology and DNA sequence data

Two new Mycetophilidae species, Neuratelia jabalmoussae sp. n. and Neuratelia salmelai sp. n. are described on the basis of material collected from Lebanon, Estonia and Finland. Detailed figures of male terminalia and photographs of general facies are provided along with discussions of their morphological distinction from sibling species. For the first time molecular characters are used to distinguish new fungus gnat species. Molecular analysis relies on cytochrome oxidase subunit one (COI) but has additionally been corroborated by information from the 28S and ITS2 regions of nuclear ribosomal DNA. Situations where morphological and molecular data provide conflicting evidence for species delimitation are discussed. A new country record from Georgia is provided for Neuratelia caucasica.     

Conjugates of 5-isoquinolinesulfonylamides and oligo-D-arginine possess high affinity and selectivity towards Rho kinase (ROCK)

In the present work, conjugates of 5-isoquinolinesulfonylamides and D-arginine-rich peptides were developed into highly potent inhibitors for basophilic protein kinases. Based on Hidaka's inhibitor H9, a generic fluorescent probe ARC-1083 was constructed possessing subnanomolar dissociation constant towards several kinases of the AGC-group. Thereafter, Hidaka's inhibitor HA1077 or Fasudil was conjugated with oligo-D-arginine resulting in the compound ARC-3002 revealing high affinity towards ROCK-II (K(d)=20 pM) and over 160-fold selectivity compared to PKAc.     

Description of Crypsiphona tasmanica sp. nov. (Lepidoptera: Geometridae: Geometrinae), with notes on limitations in using DNA barcodes for delimiting species

External morphological differences were found between Tasmanian and mainland Australian specimens of what was previously considered a single species, the Redlined Geometrid, Crypsiphona ocultaria (Donovan). Examination of genitalia showed constant differences, suggesting that Tasmanian and mainland Australian populations represent distinct species. This hypothesis was tested using DNA sequences from the mitochondrial cytochrome oxidase subunit I (COI) gene and nuclear elongation factor 1-alpha (EF-1α) gene. Tasmanian Crypsiphona Meyrick populations were found to represent a distinct species, described here as C. tasmanica sp. nov. The results show that a phylogeny-based approach allows the delimitation of C. ocultaria (Donovan) and C. tasmanica sp. nov., but distance-based delimitation is problematic due to substantial overlap in intra- and interspecific genetic distances. Using nucleotide data in character-based species delimitations might be possible for discriminating between C. ocultaria and C. tasmanica , but our current knowledge does not allow the assignment of characters required for this purpose.     

Where Do Ethno-Linguistic Groups Meet? How Copresence during Free-Time Is Related to Copresence at Home and at Work

This paper analyzes ethnic segregation across the whole activity space—at place of residence, place of work, and during free-time. We focus on interethnic meeting potential during free-time, measured as copresence, and its relationship to copresence at place of residence and work. The study is based on cellphone data for a medium-sized linguistically divided European city (Tallinn, Estonia), where the Estonian majority and mainly Russian-speaking minority populations are of roughly equal size. The results show that both places of residence and work are segregated, while other activities occur in a far more integrated environment. Copresence during free-time is positively associated with copresence at place of residence and work, however, the relationship is very weak.     

Responsive microsecond-lifetime photoluminescent probes for analysis of protein kinases and their inhibitors

Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors. Bisubstrate ARC-Lum probes bind with sub-nanomolar affinity with several PKs of the AGC group. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).