ABSTRACT
A series of methionine-proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine-proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with Ki values of 4.9 and 10.5 µM. The structure and activity relationship and the molecular docking revealed that L-proline, L-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors.
Subject(s)
Anilides/chemistry , Anilides/pharmacology , Dengue Virus , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Serine Endopeptidases/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Anilides/metabolism , Binding Sites , Catalytic Domain , Dengue Virus/drug effects , Dengue Virus/enzymology , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Humans , Methionine/chemistry , Molecular Docking Simulation , Proline/chemistry , Protease Inhibitors/metabolism , Protein Binding/drug effects , Serine Endopeptidases/metabolism , Serotyping , Stereoisomerism , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolismABSTRACT
Cone snails are predatory gastropod mollusks distributed in all tropical marine habitats with a highly sophisticated defense strategy using small peptides in their venoms. Here, we report the discovery and initial characterization of the V-superfamily conotoxins. A novel conotoxin vi15a was purified from the venom of a worm-hunting species Conus virgo. The sequence of vi15a was determined to have a unique arrangement of cysteine residues (C-C-CC-C-C-C-C), which defines the new V-superfamily conotoxins. The cDNA of vi15a was cloned with RACE method. Its unique signal peptide sequence led to the cloning of another V-superfamily conotoxin, Vt15.1, from Conus vitulinus. These results, as well as the existence of Lt15.1 from Conus litteratus and ca15a from Conus caracteristicus with the same cysteine pattern, suggest that V-superfamily might be a large and diverse group of peptides widely distributed in different Conus species. Like other eight Cys-containing toxins, V-superfamily conotoxins might also adopt an "ICK+1" disulfide bond connectivity. The identification of this novel class of conotoxins will certainly improve our understanding of the structure diversity of disulfide rich toxins.
Subject(s)
Conotoxins/chemistry , Conotoxins/classification , Conotoxins/isolation & purification , Cysteine/chemistry , Protein Sorting Signals , Amino Acid Sequence , Animals , Base Pairing , Base Sequence , Cloning, Molecular , Conotoxins/genetics , Consensus Sequence , Conus Snail/chemistry , DNA, Complementary/genetics , Disulfides/chemistry , Mass Spectrometry , Molecular Sequence Data , Species SpecificityABSTRACT
The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.