Alanine scan-guided synthesis and biological evaluation of analogues of culicinin D,a potent anticancer peptaibol

Culicinin D (1), a 10 amino acid peptaibol originally isolated from Culicinomyces clavisporus, exhibits potent activity against a range of cancer cell lines. Building on our previous work exploring the structure–activity relationship (SAR) of the unusual (2S,4S,6R)-AHMOD residue, a series of analogues of culicinin D were prepared to further investigate the SAR of these peptaibols. Alanine scanning of a potent and readily accessible analogue 23 revealed the effect of each residue on antiproliferative activity, and a small panel of analogues were prepared to explore the SAR of the non-natural amino acid residue (2S,4R)-AMD. Results from the alanine scan were used to design an expanded library of culicinin D analogues, leading to the discovery of cyclohexylalanine analogue 52, which exhibited better antiproliferative activity than the natural product 1.     

Synthesis and evaluation of conformationally constrained peptide analogues as the Src SH3 domain binding ligands

Src kinase activity is regulated by the interaction of SH3 domain with protein sequences that are rich in proline residues. Identification of more potent SH3 domain binding ligands that can regulate Src kinase activity is a subject of major interest. Conformationally constrained peptides have been previously used for improving the binding potency of the Src SH2 domain binding peptide ligands and peptide substrates of the substrate-binding site of Src. A series of peptide analogues of Ac-VSLARRPLPPLP (1, Ac-VSL12, K_d = 0.34 μM) were synthesized by introducing conformational constraints to improve the binding affinity towards the Src SH3 domain. Peptides synthesized through cyclization between N-terminal to C-terminal [VSLARRPLPPLP] or N-terminal to side chain flanking residues (i.e., [^βAVS]LARRPLPPLP and [VSLE]RRPLPPLP) exhibited at least 6.4-fold less binding affinity (K_d = 2.19–4.85 μM) when compared to 1. The data suggest upon N-terminal cyclization with C-terminal or flanking residues, the interactions of the amino acids in the core RPLPPLP reduce significantly with the residues within the Src SH3 domain. Conformationally constrained peptide V[SLARRPLPPLP] (5) was synthesized through cyclization of C-terminal to the serine side chain and displayed a comparable binding affinity (K_d = 0.35 μM) towards the Src SH3 domain versus that of 1. Thus, this template may be used to optimize and generate more potent analogues with higher stability.     

Total Synthesis of Septocylindrin B and C-Terminus Modified Analogues

The total synthesis is reported of the peptaibol Septocylindrin B which is related to the well documented channel forming peptaibol antibiotic Alamethicin. Several analogues were synthesized with a modified C-terminus, to investigate the SAR of the terminal residue Phaol. All these peptides were tested for their membrane perturbation properties by fluorescent dye leakage assay and for their antibacterial activity.     

The amino acid sequence of plastocyanin from French bean (Phaseolus vulgaris)

The amino acid sequence of the plastocyanin from French bean (Phaseolus vulgaris) was determined. The protein consists of a single polypeptide chain of 99 residues, and the sequence was determined by characterization of CNBr, tryptic, chymotryptic and thermolysin peptides. When the sequence is compared with that from the plastocyanin of the unicellular green alga Chlorella fusca, the French-bean protein shows the deletion of the N-terminal residue, a two residue insertion and 53 identical residues. Detailed evidence for the sequence of the protein has been deposited as Supplementary Publication SUP 50037 (16pp., 1 microfiche) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Anti-HAV evaluation and molecular docking of newly synthesized 3-benzyl(phenethyl)benzo[g]quinazolines

Synthesized 3-benzyl(phenethyl)benzo[g]quinazolines (1–17) were evaluated in vitro to determine their effects against the anti-hepatitis A virus (HAV) using a cytopathic effect inhibition assay. Of the synthesized compounds, 16 and 17 showed considerably high anti-HAV activity, as indicated by their EC₅₀ values of 27.59 and 18 μM, respectively, when compared to that of amantadine (37.3 μM), the standard therapeutic agent. In addition, they exhibited low cytotoxicity as indicated by their CC₅₀ values, 290.63 and 569.45 μM, respectively. Compounds 1, 2, and 5 exhibited remarkable activity compared to the active compounds (16, 17) and amantadine. The selectivity index (SI) values were calculated and applied as a parameter for classifying the activity of the targets. In addition, molecular docking was performed to rationalize the SAR of the target compounds and analyze the binding modes between the docked-selected compounds and amino acid residues in the active site of the HAV-3C proteinase enzyme.     

Second-generation derivatives of the eukaryotic translation initiation inhibitor pateamine A targeting eIF4A as potential anticancer agents

A series of pateamine A (1) derivatives were synthesized for structure/activity relationship (SAR) studies and a selection of previous generation analogs were re-evaluated based on current information regarding the mechanism of action of these translation inhibitors. Structural modifications in the new generation of derivatives focused on alterations to the C19–C22 Z,E-diene and the trienyl side chain of the previously described simplified, des-methyl, des-amino pateamine A (DMDAPatA, 2). Derivatives were tested for anti-proliferative activity in cell culture and for inhibition of mammalian cap-dependent translation in vitro. Activity was highly dependent on the rigidity and conformation of the macrolide and the functionality of the side chain. The only well tolerated substitutions were replacement of the N,N-dimethyl amino group found on the side chain of 2 with other tertiary amine groups. SAR reported here suggests that this site may be modified in future studies to improve serum stability, cell-type specificity, and/or specificity towards rapidly proliferating cells.     

Design and synthesis of novel antimicrobials with activity against Gram-positive bacteria and mycobacterial species,including M. tuberculosis

The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereospecific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC) assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Based on these studies the acrylic ester 20 has been developed as a potential lead compound which was found to have an MIC value of 0.4 μg/mL against Mycobacterium tuberculosis. The SAR and biological activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent activity of this series of analogs.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

Identification of novel peptidomimetics targeting the polo-box domain of polo-like kinase 1

A series of new peptidomimetics targeting the polo-box domain (PBD) of polo-like kinase 1 (Plk1) was identified based on the potent and selective pentapeptide Plk1 PBD inhibitor PLHSpT. Unnatural amino acid residues were introduced to the newly designed compound and the N-terminal substituent of the peptidomimetic was investigated. The optimized compound 9 inhibited the Plk1 PBD with IC₅₀ of 0.267 μM and showed almost no inhibition to Plk2 PBD or Plk3 PBD at 100 μM. Biolayer interferometry studies demonstrated that compound 9 showed potent binding affinity to Plk1 with a K_d value of 0.164 μM, while no K_d were detected against Plk2 and Plk3. Compound 9 showed improved stability in rat plasma compared to PLHSpT. Binding mode analysis was performed and in agreement with the observed experimental results. There are only two natural amino acids remained in the chemical structure of 9. This study may provide new information for further research on Plk1 PBD inhibitors.     

Development of an efficient semisynthetic modification of FR901459 via a novel regioselective N,O-acyl migration

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.     

Effect of the C-terminal domains and terminal residues of catalytic domain on enzymatic activity and thermostability of lichenase from Clostridium thermocellum

To elucidate the effects of C-terminal domains of LicMB (mature lichenase from Clostridium thermocellum) and terminal residues of LicMB-CD (catalytic domain of LicMB) on the properties of lichenase, a series of truncated genes were constructed and expressed in E. coli. The Thr-Pro box had a positive effect while the dockerin domain had a negative impact on the properties of LicMB. The N-terminal 10–25th and C-terminal 1–9th residues of LicMB-CD were necessary to retain high thermostability while the N-terminal 1–7th and C-terminal 1–3rd residues were not necessary to maintain enzymatic activity.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Design,synthesis and biological evaluation of N,N-3-phenyl-3-benzylaminopropanamide derivatives as novel cholesteryl ester transfer protein inhibitor

A series of N,N-3-phenyl-3-benzylaminopropanamide derivatives were identified as novel CETP (cholesteryl ester transfer protein) inhibitors. In our previous study, lead compound L10 was discovered by pharmacophore-based virtual screening (Dong-Mei Zhao et al., 2014). Based on L10 (IC₅₀ 8.06 μM), compound HL6 (IC₅₀ 10.7 μM) was discovered following systematic structure variation and biological tests. Further optimization of the structure–activity relationship (SAR) resulted in N,N-3-phenyl-3-benzylaminopro panamides derivatives as novel CETP inhibitors. They were synthesized and evaluated against CETP by BODIPY-CE fluorescence assay. Among them, HL16 (IC₅₀ 0.69 μM) was a highly potent CETP inhibitor in vitro. In addition, HL16 exhibited favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. The molecular docking of HL16 into the CETP was performed. The binding mode demonstrated that HL16 occupied the CETP binding site and formed interactions with the key amino acid residues.     

Discovery of heterocyclic replacements for the coumarin core of anti-tubercular FadD32 inhibitors

Previous work established a coumarin scaffold as a starting point for inhibition of Mycobacterium tuberculosis (Mtb) FadD32 enzymatic activity. After further profiling of the coumarin inhibitor 4 revealed chemical instability, we discovered that a quinoline ring circumvented this instability and had the advantage of offering additional substitution vectors to further optimize. Ensuing SAR studies gave rise to quinoline-2-carboxamides with potent anti-tubercular activity. Further optimization of ADME/PK properties culminated in 21b that exhibited compelling in vivo efficacy in a mouse model of Mtb infection.     

Improvement of both plasmepsin inhibitory activity and antimalarial activity by 2-aminoethylamino substitution

We attached 2-aminoethylamino groups to allophenylnorstatine-containing plasmepsin (Plm) inhibitors and investigated SAR of the methyl or ethyl substitutions on the amino groups. Unexpectedly, compounds 22 (KNI-10743) and 25 (KNI-10742) exhibited extremely potent Plm II inhibitory activities (K_i <0.1 nM). Moreover, among our peptidomimetic Plm inhibitors, we identified the compounds with the highest antimalarial activity using a SYBR Green I-based fluorescence assay.     

Urotensin core mimics that modulate the biological activity of urotensin-II related peptide but not urotensin-II

A novel approach for the synthesis of head-to-tail cyclic peptides has been developed and used to prepare two mimics of the urotensin II-related peptide (URP) cyclic core. Mimics 1 and 2 (c[Trp-Lys-Tyr-Gly-ψ(triazole)-Gly] and c[Phe-Trp-Lys-Tyr-Gly-ψ(triazole)-Gly]) were respectively prepared using a combination of solid- and solution-phase synthesis. The silyl-based alkyne-modifying (SAM) linker enabled installation of C-terminal alkyne and N-terminal azide moieties onto linear peptide precursors, which underwent head-to-tail copper-catalyzed azide-alkyne cycloaddition (CuAAC) in solution. In an aortic ring contraction assay, neither 1 nor 2 exhibited agonist activity; however, both inhibited selectively URP- but not UII-mediated vasoconstriction. The core phenylalanine residue was shown to be important for enhancing modulatory activity of the urotensinergic system.     

Characterization of lamprey fibrinopeptides

1. Lamprey fibrinopeptide B is a relatively large peptide made up of about 40 amino acid residues. The peptide is highly electronegative, containing a large number of aspartic acid residues and a tyrosine O-sulphate residue. 2. The amino acid sequence of the first 18 residues from the N-terminal end of fibrinopeptide B has been established. The C-terminal ends with the sequence Val-Arg. Fibrino-peptide B is released by both lamprey and bovine thrombins. 3. Lamprey fibrino-peptide A is a short peptide containing only eight residues. The proposed amino acid sequence is: Asp-Asp-Ser-Ile/Leu-Asp-Ser-Leu/Ile-ArgThis peptide is released by lamprey thrombin but not by bovine thrombin.     

Characterization of the peptidases of Calliphora: Many features allow the utilization of small peptides as an amino acid reservoir

Electrophoretic separation of whole flies and of haemolymph indicates the presence of four peptidases, named dipeptidase A, B and C (Dip A, B and C) and leucine amino peptidase (LAP) after enzymes of similar substrate specificities and electrophoretic mobilities found in Drosophila (Laurie-Ahlberg, Biochem. Genet.20, 407–424, 1982; Walker et al., Insect Biochem.10, 535–541, 1980). Prominent in both tissues and haemolymph, dipeptidase A and B together hydrolyse a variety of dipeptides in vitro and probably most of the fly's small peptide component in vivo. Though Dip A and Dip B hydrolyse many of the same substrates, their activities differ in at least several respects. Dip A's K_ms are higher than Dip B's K_ms and hence in vivo the two enzymes together are likely to provide peptide hydrolysis through a wide range of substrate concentration. Dip A's unique hydrolyses are of peptides with biosynthesized amino acids in the N-terminal position and Dip B's unique hydrolyses are of peptides with essential amino acids in the N-terminal position. Dip B, but not Dip A, is inhibited by free amino acid. It is inhibited non-competitively and most strongly by essential amino acids. In cell-free haemolymph Dip B's activity is more stable than Dip A's. The accumulation and maintenance of small peptides in times of dietary sufficiency and the utilization of the small peptides as a source of amino acid in times of dietary scarcity (Collett, Insect Biochem.6, 179–185, 1976a; J. Insect Physiol.22, 1433–1440, 1976b) may be attributed to these features.     

Rational design of bis-indolylmethane-oxadiazole hybrids as inhibitors of thymidine phosphorylase

Inhibition of Thymidine phosphorylase (TP) is continuously studied for the design and development of new drugs for the treatment of neoplastic diseases. As a part of our effort to identify TP inhibitors, we performed a structure-based virtual screening (SBVS) of our compound collection. Based on the insights gained from structures of virtual screening hits, a scaffold was designed using 1,3,4-oxadiazole as the basic structural feature and SAR studies were carried out for the optimization of this scaffold. Twenty-five novel bis-indole linked 1,3,4-oxadiazoles (7–31) were designed, synthesized and tested in vitro against E. coli TP (EcTP). Compound 7 emerged as potent TP inhibitor with an IC₅₀ value of 3.50?±?0.01?μM. Docking studies were carried out using GOLD software on thymidine phosphorylase from human (hTP) and E. coli (EcTP). Various hydrogen bonding, hydrophobic interactions, and π-π stacking were observed between designed molecules and the active site amino acid residues of the studied enzymes.     

Design and synthesis of novel thiadiazole-thiazolone hybrids as potential inhibitors of the human mitotic kinesin Eg5

A novel series of 1,3,4-thiadiazole-thiazolone hybrids 5a–v were designed, synthesized, characterized, and evaluated against the basal and the microtubule (MT)-stimulated ATPase activity of Eg5. From the evaluated derivatives, 5h displayed the highest inhibition with an IC₅₀ value of 13.2?µM against the MT-stimulated Eg5 ATPase activity. Similarly, compounds 5f and 5i also presented encouraging inhibition with IC₅₀ of 17.2?µM and 20.2?µM, respectively. A brief structure–activity relationship (SAR) analysis indicated that 2-chloro and 4-nitro substituents on the phenyl ring of the thiazolone motif contributed significantly to enzyme inhibition. An in silico molecular docking study using the crystal structure of Eg5 further supported the SAR and reasoned the importance of crucial molecular protein–ligand interactions in influencing the inhibition of the ATPase activity of Eg5. The magnitude of the electron-withdrawing functionalities over the hybrids and the critical molecular interactions contributed towards higher in vitro potency of the compounds. The drug-like properties of the synthesized compounds 5a–v were also calculated based on the Lipinski’s rule of five and in silico computation of key pharmacokinetic parameters (ADME). Thus, the present work unveils these hybrid molecules as novel Eg5 inhibitors with promising drug-like properties for future development.