Ranacyclin-NF, a Novel Bowman–Birk Type Protease Inhibitor from the Skin Secretion of the East Asian Frog, Pelophylax nigromaculatus

Serine protease inhibitors are found in plants, animals and microorganisms, where they play important roles in many physiological and pathological processes. Inhibitor scaffolds based on natural proteins and peptides have gradually become the focus of current research as they tend to bind to their targets with greater specificity than small molecules. In this report, a novel Bowman–Birk type inhibitor, named ranacyclin-NF (RNF), is described and was identified in the skin secretion of the East Asian frog, Pelophylax nigromaculatus. A synthetic replicate of the peptide was subjected to a series of functional assays. It displayed trypsin inhibitory activity with an inhibitory constant, Ki, of 447 nM and had negligible direct cytotoxicity. No observable direct antimicrobial activity was found but RNF improved the therapeutic potency of Gentamicin against Methicillin-resistant Staphylococcus aureus (MRSA). RNF shared significant sequence similarity to previously reported and related inhibitors from Odorrana grahami (ORB) and Rana esculenta (ranacyclin-T), both of which were found to be multi-functional. Two analogues of RNF, named ranacyclin-NF1 (RNF1) and ranacyclin-NF3L (RNF3L), were designed based on some features of ORB and ranacyclin-T to study structure–activity relationships. Structure–activity studies demonstrated that residues outside of the trypsin inhibitory loop (TIL) may be related to the efficacy of trypsin inhibitory activity.

Download Full-text

Structure–Activity Relationship and Molecular Docking of a Kunitz-Like Trypsin Inhibitor, Kunitzin-AH, from the Skin Secretion of Amolops hainanensis

Pharmaceutics ◽

10.3390/pharmaceutics13070966 ◽

2021 ◽

Vol 13 (7) ◽

pp. 966

Author(s):

Yuqing Chen ◽

Xinping Xi ◽

Chengbang Ma ◽

Mei Zhou ◽

Xiaoling Chen ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Inhibitory Activity ◽

Structure Activity Relationship ◽

Activity Relationship ◽

Skin Secretion ◽

Site Mutation ◽

Structure Activity ◽

Hydrophilic Residues ◽

Inhibitory Drug ◽

Relationship Of

Kunitz-like trypsin inhibitors are one of the most noteworthy research objects owing to their significance in pharmacological studies, including anticarcinogenic activity, obesity regulation and anticoagulation. In the current study, a novel Kunitz-like trypsin inhibitor, Kunitzin-AH, was isolated from the skin secretion of Amolops hainanensis. The novel peptide displayed a modest trypsin inhibitory activity with the inhibitor constant (Ki) value of 1.18 ± 0.08 µM without inducing damage to healthy horse erythrocytes. Then, a series of shortened variants of Kunitzin-AH were designed by truncating a peptide loop and site mutation inside the loop to illustrate the structure–activity relationship of the trypsin inhibition function. Among the variants, a significant decrease was observed for the Cys-Cys loop domain, while the extension of an Arg at N-terminus (RCKAAFC) retained the inhibitory activity, indicating that the -RCK-motif is essential in forming the reactive domain for exerting the inhibitory activity. Furthermore, substitutions of Ala by hydrophobic or hydrophilic residues decreased the activity, indicating suitable steric hindrance provides convenience for the combination of trypsin. Additionally, the conformational simulation of the analogues processed with Chimera and Gromacs and further combination simulations between the peptides and trypsin conducted with HDOCK offered a potential opportunity for the natural trypsin inhibitory drug design. The truncated sequence, AH-798, may be a good replacement for the full-length peptide, and can be optimized via cyclization for further study.

Download Full-text

A Novel Kunitzin-Like Trypsin Inhibitor Isolated from Defensive Skin Secretion of Odorrana versabilis

Biomolecules ◽

10.3390/biom9070254 ◽

2019 ◽

Vol 9 (7) ◽

pp. 254 ◽

Cited By ~ 1

Author(s):

Yanjing Dong ◽

Daning Shi ◽

Yuan Ying ◽

Xinping Xi ◽

Xiaoling Chen ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Inhibitory Activity ◽

The Novel ◽

Skin Secretion ◽

Amphibian Skin ◽

Sequence Alignments ◽

Molecular Weights ◽

Trypsin Inhibitory Activity ◽

Skin Secretions ◽

Potent Inhibitory Activity

Protease inhibitors that were identified from amphibian skin secretions with low molecular weights and potent inhibitory activity were thought to be potential candidates for novel peptide drugs. Here, a novel peptide with trypsin inhibitory activity was found in the skin secretion of the Chinese bamboo leaf odorous frog, Odorrana versabilis. Based on the sequence alignments of sequencing results, the novel peptide (ALKYPFRCKAAFC) was named as Kunitzin-OV. The synthetic replicate of Kunitzin-OV was subjected to a series of functional assays, and it exhibited a trypsin inhibitory activity with a Ki value of 3.042 µM, whereas, when Lys-9 at P1 position was substituted by Phe, trypsin inhibitory activity was undetected and the chymotrypsin inhibitory activity was optimized with a Ki value of 2.874 µM. However, its protease-binding loop was catabolized by trypsin during the trypsin cleavage test. In conclusion, Kunizin-OV is a novel peptide with trypsin inhibitory activity as a member of kunitzins, which is a non-typical Kunitz-like trypsin inhibitor with a highly conserved reactive site (K-A) and quite a short sequence.

Download Full-text

Independent pseudogenizations and losses of sox15 during amniote diversification following asymmetric ohnolog evolution

BMC Ecology and Evolution ◽

10.1186/s12862-021-01864-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yusaku Ogita ◽

Kei Tamura ◽

Shuuji Mawaribuchi ◽

Nobuhiko Takamatsu ◽

Michihiko Ito

Keyword(s):

Sequence Similarity ◽

The Other ◽

Vertebrate Evolution ◽

Skeletal Muscle Regeneration ◽

Cns Development ◽

Placental Development ◽

Relaxed Selection ◽

Significant Sequence Similarity ◽

Anuran Amphibians ◽

Divergent Gene

Abstract Background Four ohnologous genes (sox1, sox2, sox3, and sox15) were generated by two rounds of whole-genome duplication in a vertebrate ancestor. In eutherian mammals, Sox1, Sox2, and Sox3 participate in central nervous system (CNS) development. Sox15 has a function in skeletal muscle regeneration and has little functional overlap with the other three ohnologs. In contrast, the frog Xenopus laevis and zebrafish orthologs of sox15 as well as sox1-3 function in CNS development. We previously reported that Sox15 is involved in mouse placental development as neofunctionalization, but is pseudogenized in the marsupial opossum. These findings suggest that sox15 might have evolved with divergent gene fates during vertebrate evolution. However, knowledge concerning sox15 in other vertebrate lineages than therian mammals, anuran amphibians, and teleost fish is scarce. Our purpose in this study was to clarify the fate and molecular evolution of sox15 during vertebrate evolution. Results We searched for sox15 orthologs in all vertebrate classes from agnathans to mammals by significant sequence similarity and synteny analyses using vertebrate genome databases. Interestingly, sox15 was independently pseudogenized at least twice during diversification of the marsupial mammals. Moreover, we observed independent gene loss of sox15 at least twice during reptile evolution in squamates and crocodile-bird diversification. Codon-based phylogenetic tree and selective analyses revealed an increased dN/dS ratio for sox15 compared to the other three ohnologs during jawed vertebrate evolution. Conclusions The findings revealed an asymmetric evolution of sox15 among the four ohnologs during vertebrate evolution, which was supported by the increased dN/dS values in cartilaginous fishes, anuran amphibians, and amniotes. The increased dN/dS value of sox15 may have been caused mainly by relaxed selection. Notably, independent pseudogenizations and losses of sox15 were observed during marsupial and reptile evolution, respectively. Both might have been caused by strong relaxed selection. The drastic gene fates of sox15, including neofunctionalization and pseudogenizations/losses during amniote diversification, might be caused by a release from evolutionary constraints.

Download Full-text

Discriminating between JCPyV and BKPyV in Urinary Virome Data Sets

Viruses ◽

10.3390/v13061041 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1041

Author(s):

Rita Mormando ◽

Alan J. Wolfe ◽

Catherine Putonti

Keyword(s):

Jc Virus ◽

Sequence Similarity ◽

Bk Virus ◽

Data Sets ◽

Metagenomic Sequencing ◽

Significant Sequence Similarity ◽

Sequence Identity ◽

Shotgun Metagenomic Sequencing ◽

Urinary Microbiome ◽

Six Genes

Polyomaviruses are abundant in the human body. The polyomaviruses JC virus (JCPyV) and BK virus (BKPyV) are common viruses in the human urinary tract. Prior studies have estimated that JCPyV infects between 20 and 80% of adults and that BKPyV infects between 65 and 90% of individuals by age 10. However, these two viruses encode for the same six genes and share 75% nucleotide sequence identity across their genomes. While prior urinary virome studies have repeatedly reported the presence of JCPyV, we were interested in seeing how JCPyV prevalence compares to BKPyV. We retrieved all publicly available shotgun metagenomic sequencing reads from urinary microbiome and virome studies (n = 165). While one third of the data sets produced hits to JCPyV, upon further investigation were we able to determine that the majority of these were in fact BKPyV. This distinction was made by specifically mining for JCPyV and BKPyV and considering uniform coverage across the genome. This approach provides confidence in taxon calls, even between closely related viruses with significant sequence similarity.

Download Full-text

Synthesis of oxadiazole-2-oxide derivatives as potential drug candidates for schistosomiasis targeting SjTGR

Parasites & Vectors ◽

10.1186/s13071-021-04634-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Gongming Li ◽

Qingqing Guo ◽

Chao Feng ◽

Huan Chen ◽

Wenjiao Zhao ◽

...

Keyword(s):

Schistosoma Japonicum ◽

Inhibitory Activity ◽

Pyridine Ring ◽

New Drugs ◽

Wild Type ◽

Drug Candidates ◽

Structure Activity ◽

Docking Method ◽

Antigen Protein ◽

Thioredoxin Glutathione Reductase

Abstract Background Schistosomiasis is a chronic parasitic disease that affects millions of people’s health worldwide. Because of the increasing drug resistance to praziquantel (PZQ), which is the primary drug for schistosomiasis, developing new drugs to treat schistosomiasis is crucial. Oxadiazole-2-oxides have been identified as potential anti-schistosomiasis reagents targeting thioredoxin glutathione reductase (TGR). Methods In this work, one of the oxadiazole-2-oxides derivatives furoxan was used as the lead compound to exploit a series of novel furoxan derivatives for studying inhibitory activity against both recombinant Schistosoma japonicum TGR containing selenium (rSjTGR-Sec) and soluble worm antigen protein (SWAP) containing wild-type Schistosoma japonicum TGR (wtSjTGR), in order to develop a new leading compound for schistosomiasis. Thirty-nine novel derivatives were prepared to test their activity toward both enzymes. The docking method was used to detect the binding site between the active molecule and SjTGR. The structure–activity relationship (SAR) of these novel furoxan derivatives was preliminarily analyzed. Results It was found that several new derivatives, including compounds 6a–6d, 9ab, 9bd and 9be, demonstrated greater activity toward rSjTGR-Sec or SWAP containing wtSjTGR than did furoxan. Interestingly, all intermediates bearing hydroxy (6a–6d) showed excellent inhibitory activity against both enzymes. In particular, compound 6d with trifluoromethyl on a pyridine ring was found to have much higher inhibition toward both rSjTGR-Sec (half-maximal inhibitory concentration, IC50,7.5nM) and SWAP containing wtSjTGR (IC50 55.8nM) than furoxan. Additionally, the docking method identified the possible matching sites between 6d and Schistosoma japonicum TGR (SjTGR), which theoretically lends support to the inhibitory activity of 6d. Conclusion The data obtained herein showed that 6d with trifluoromethyl on a pyridine ring could be a valuable leading compound for further study.

Download Full-text

Active fragments obtained by cyanogen bromide cleavage of ovomucoid

Biochemical Journal ◽

10.1042/bj1550345 ◽

1976 ◽

Vol 155 (2) ◽

pp. 345-351 ◽

Cited By ~ 12

Author(s):

J G. Beeley

Keyword(s):

Molecular Weight ◽

Trypsin Inhibitor ◽

Inhibitory Activity ◽

Cyanogen Bromide ◽

Peptide Chain ◽

Terminal Portion ◽

Trypsin Inhibitory Activity ◽

Methionine Residues ◽

Active Fragments

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.

Download Full-text

ChemInform Abstract: Structure-Activity Relationships of Biflavonoids for β-Secretase (BACE-1) Inhibitory Activity.

ChemInform ◽

10.1002/chin.201311145 ◽

2013 ◽

Vol 44 (11) ◽

pp. no-no

Author(s):

Hiroaki Sasaki ◽

Yuki Kitoh ◽

Kazuhiko Miki ◽

Kaoru Kinoshita ◽

Kiyotaka Koyama ◽

...

Keyword(s):

Inhibitory Activity ◽

Structure Activity Relationships ◽

Structure Activity ◽

Abstract Structure ◽

Bace 1

Download Full-text

An intergenic G-rich region in Leishmania tarentolae kinetoplast maxicircle DNA is a pan-edited cryptogene encoding ribosomal protein S12

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.56-67.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 56-67

Author(s):

D A Maslov ◽

N R Sturm ◽

B M Niner ◽

E S Gruszynski ◽

M Peris ◽

...

Keyword(s):

Amino Acid ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Sequence Similarity ◽

Rich Region ◽

Leishmania Tarentolae ◽

Significant Sequence Similarity ◽

The Family ◽

Intergenic Regions ◽

Ribosomal Protein S12

Six short G-rich intergenic regions in the maxicircle of Leishmania tarentolae are conserved in location and polarity in two other kinetoplastid species. We show here that G-rich region 6 (G6) represents a pan-edited cryptogene which contains at least two domains edited independently in a 3'-to-5' manner connected by short unedited regions. In the completely edited RNA, 117 uridines are added at 49 sites and 32 uridines are deleted at 13 sites, creating a translated 85-amino-acid polypeptide. Similar polypeptides are probably encoded by pan-edited G6 transcripts in two other species. The G6 polypeptide has significant sequence similarity to the family of S12 ribosomal proteins. A minicircle-encoded gRNA overlaps 12 editing sites in G6 mRNA, and chimeric gRNA/mRNA molecules were shown to exist, in agreement with the transesterification model for editing.

Download Full-text