Rhomboid proteases form one of the most widespread intramembrane protease families. They have been implicated in variety of human diseases. The currently reported rhomboid inhibitors display some selectivity, but their construction involves multistep synthesis protocols. Here, we report benzoxazin-4-ones as novel inhibitors of rhomboid proteases with a covalent, but slow reversible inhibition mechanism. Benzoxazin-4-ones can be synthesized from anthranilic acid derivatives in a one-step synthesis, making them easily accessible. We demonstrate that an alkoxy substituent at the 2-position is crucial for potency and results in low micromolar inhibitors of rhomboid proteases. Hence, we expect that these compounds will allow rapid synthesis and optimization of inhibitors of rhomboids from different organisms.

• MeSH
• Bacillus subtilis enzymologie   MeSH
• benzoxaziny chemická syntéza   chemie   farmakologie   MeSH
• chymotrypsin antagonisté a inhibitory   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   MeSH
• endopeptidasy MeSH
• enzymatické testy MeSH
• Escherichia coli enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   farmakologie   MeSH
• inhibitory trypsinu chemická syntéza   chemie   farmakologie   MeSH
• membránové proteiny antagonisté a inhibitory   MeSH
• molekulární struktura MeSH
• ortoaminobenzoáty chemie   MeSH
• proteiny z Escherichia coli antagonisté a inhibitory   MeSH
• skot MeSH
• trypsin chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. RESULTS: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed. CONCLUSION: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.

• MeSH
• DNA helmintů chemie   MeSH
• fylogeneze MeSH
• infekce červy třídy Trematoda parazitologie   veterinární   MeSH
• inhibitory serinových proteinas chemie   genetika   izolace a purifikace   metabolismus   MeSH
• kapři parazitologie   MeSH
• nemoci ryb parazitologie   MeSH
• počítačová simulace MeSH
• polymerázová řetězová reakce MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• serpiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Trematoda chemie   klasifikace   enzymologie   genetika   MeSH
• žábry parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel rhomboid inhibitor scaffold with ample opportunity for optimization.

• MeSH
• benzoxaziny chemická syntéza   chemie   MeSH
• chymotrypsin chemie   MeSH
• DNA vazebné proteiny antagonisté a inhibitory   chemie   genetika   MeSH
• Drosophila chemie   MeSH
• endopeptidasy chemie   genetika   MeSH
• enzymatické testy MeSH
• Escherichia coli enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   MeSH
• katalytická doména MeSH
• lidé MeSH
• membránové proteiny antagonisté a inhibitory   chemie   genetika   MeSH
• mutace MeSH
• objevování léků MeSH
• proteiny Drosophily metabolismus   MeSH
• proteiny z Escherichia coli antagonisté a inhibitory   chemie   genetika   MeSH
• serin chemie   MeSH
• simulace molekulového dockingu MeSH
• skot MeSH
• styreny chemická syntéza   chemie   MeSH
• transformující růstový faktor alfa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Rhomboids are intramembrane serine proteases and belong to the group of structurally and biochemically most comprehensively characterized membrane proteins. They are highly conserved and ubiquitously distributed in all kingdoms of life and function in a wide range of biological processes, including epidermal growth factor signaling, mitochondrial dynamics, and apoptosis. Importantly, rhomboids have been associated with multiple diseases, including Parkinson's disease, type 2 diabetes, and malaria. However, despite a thorough understanding of many structural and functional aspects of rhomboids, potent and selective inhibitors of these intramembrane proteases are still not available. In this study, we describe the computer-based rational design, chemical synthesis, and biological evaluation of novel N-methylene saccharin-based rhomboid protease inhibitors. Saccharin inhibitors displayed inhibitory potency in the submicromolar range, effectiveness against rhomboids both in vitro and in live Escherichia coli cells, and substantially improved selectivity against human serine hydrolases compared to those of previously known rhomboid inhibitors. Consequently, N-methylene saccharins are promising new templates for the development of rhomboid inhibitors, providing novel tools for probing rhomboid functions in physiology and disease.

• MeSH
• design s pomocí počítače MeSH
• HEK293 buňky MeSH
• inhibitory serinových proteinas chemie   farmakologie   MeSH
• lidé MeSH
• membránové proteiny MeSH
• racionální návrh léčiv * MeSH
• sacharin analogy a deriváty   farmakologie   MeSH
• serinové proteasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

• MeSH
• gramnegativní bakterie enzymologie   MeSH
• grampozitivní bakterie enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   farmakologie   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• proteasy metabolismus   MeSH
• racionální návrh léčiv * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Prolyl oligopeptidase (POP), also called prolyl endopeptidase, is a cytosolic enzyme investigated by several research groups. It has been proposed to play an important role in physiological processes such as modulation of the levels of several neuronal peptides and hormones containing a proline residue. Due to its proteolytic activity and physiological role in cell signaling pathways, inhibition of POP offers an emerging approach for the treatment of Alzheimer's and Parkinson's diseases as well as other diseases related to cognitive impairment. Furthermore, it may also represent an interesting target for treatment of neuropsychiatric disorders, and as an antiangiogenesis or antineoplastic agent. In this review paper, we summarized naturally occurring POP inhibitors together with peptide-like inhibitors and their biological effects. Some of them have shown promising results and interesting pharmacological profiles. However, to date, there is no POP inhibitor available on the market although several clinical trials have been undertaken.

• MeSH
• inhibitory serinových proteinas chemie   farmakologie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• serinové endopeptidasy metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyses proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. Thirty-one isoquinoline alkaloids of various structural types, previously isolated in our laboratory, were screened for their ability to inhibit prolyl oligopeptidase. Promising results have been showed by alkaloids californidine (IC50=55.6±3.5 μM), dihydrosanquinarine (IC50=99.1±7.6 μM), corypalmine (IC50=128.0±10.5 μM) and N-methyllaurotetanine (IC50=135.0±11.7 μM).

• MeSH
• alkaloidy chemie   MeSH
• aporfiny chemie   MeSH
• dioxoly chemie   MeSH
• heterocyklické sloučeniny tetra- a více cyklické chemie   MeSH
• inhibitory serinových proteinas chemie   MeSH
• isochinoliny chemie   MeSH
• molekulární struktura MeSH
• serinové endopeptidasy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions. RESULTS: We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for β-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for β-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases). CONCLUSIONS: By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level.

• MeSH
• aminokyselinové motivy MeSH
• cystein chemie   genetika   MeSH
• fylogeneze MeSH
• inhibitory serinových proteinas chemie   klasifikace   genetika   metabolismus   MeSH
• klíště chemie   genetika   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• proteiny členovců chemie   klasifikace   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   klasifikace   genetika   metabolismus   MeSH
• Rhipicephalus chemie   genetika   metabolismus   MeSH
• sekvenční homologie aminokyselin MeSH
• slinné proteiny a peptidy chemie   klasifikace   genetika   metabolismus   MeSH
• terciární struktura proteinů MeSH
• tryptasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• inhibitory serinových proteinas chemie   MeSH
• nemoc MeSH
• serinové endopeptidasy chemie   MeSH
• serpiny biosyntéza   chemie   MeSH
• zdraví MeSH