Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2

Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped k_{cat,app – acetyl-CoA} and (k_cat/K_m)_{app – acetyl-CoA} pH-rate profiles identified two apparent pK_a,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the k_cat,app value nor did it abolish the pK_a,app value attributed to the general base in catalysis (pK_a ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis.     

Exploiting the 7-methylimidazo[1,5-a]pyrazin-8(7H)-one scaffold for the development of novel chemical inhibitors for Bromodomain and Extraterminal Domain (BET) family

The bromodomain and extraterminal (BET) family of proteins play a crucial role in promoting gene expression of critical oncogenes. Novel BET bromodomain inhibitors with excellent potency, drug metabolism and pharmacokinetics (DMPK) properties were in strong need for development. We reported a series of potential BET inhibitors through incorporation of imidazole into pyridine scaffold. Among them, a novel BET inhibitor with 7-methylimidazo[1,5-a]pyrazin-8(7H)-one core, compound 28, was considered to be the most promising for in-depth study. Compound 28 exhibited excellent BRD4-inhibitory activity with IC₅₀ value of 33 nM and anti-proliferation potency with IC₅₀ value of 110 nM in HL-60 (human promyelocytic leukemia) cancer cell lines. Western Blot indicated that compound 28 can effectively trigger apoptosis in BxPc3 cells by modulating the intrinsic apoptotic pathway. In conclusion, these results suggested that compound 28 has merely potential for leukemia treatment.     

Promising antibacterial agents against multidrug resistant Staphylococcus aureus

Rapid emergence of multidrug resistant Staphylococcus aureus infections has created a critical health menace universally. Resistance to all the available chemotherapeutics has been on rise which led to WHO to stratify Staphylococcus aureus as high tier priorty II pathogen. Hence, discovery and development of new antibacterial agents with new mode of action is crucial to address the multidrug resistant Staphylococcus aureus infections. The egressing understanding of new antibacterials on their biological target provides opportunities for new therapeutic agents. This review underlines on various aspects of drug design, structure activity relationships (SARs) and mechanism of action of various new antibacterial agents and also covers the recent reports on new antibacterial agents with potent activity against multidrug resistant Staphylococcus aureus. This review provides attention on in vitro and in vivo pharmacological activities of new antibacterial agents in the point of view of drug discovery and development.     

Imidazolo and tryptophan-imidazolo hybrid derived ureas/thioureas as potent bioactive agents – SAR and molecular modelling studies

Herein, we used an imidazole derivative (IMD) which showed promising antibacterial, antifungal and antioxidant properties in our earlier investigation. Prompted by this, we converted IMD to hydrazide (IMH) by hydrazinolysis which was derivatized to various ureas (3–7) and thioureas (8–12). On the other hand, IMH was conjugated to Boc-Trp-OH as it has been shown in the past that hybridization of two molecules produced promising biologically active compounds. Boc of the conjugate was removed and further converted into several urea (14–18) and thiourea (19–23) derivatives. All the title compounds so also the starting materials and intermediates were assessed for potential biological applications. The results showed that compounds 3, 4, 8, 9, 14, 15, 19 and 20 were excellent antioxidants as revealed by DPPH, DMPD and ABTS assays. Further, certain analogues like 5–7, 10–12, 16–18 and 21–23 were found to be potent antimicrobials against pathogenic bacteria and fungi whereas good anti-inflammatory activity was obtained for molecules 5–7, 10–12, 16–18 and 21–23. All together, derivatives of the conjugates have shown superior activity over non-conjugated compounds and the former have exhibited potent activity against standard drugs in all the assays. In a quest to understand the binding interactions of the compounds with active site of tyrosine kinase (PDB ID: 2HCK), glucosamine-6-phosphate (GlcN-6-P) synthase (PDB ID: 2VF5) and cyclooxygenase-2 (PDB ID: 1CX2) enzymes, the correlation studies were conducted using molecular modelling which showed good receptor binding interactions with several amino acids of the enzymes. Overall, the current investigation may be considered for the discovery of lead compound(s) for treating multiple disorder conditions using singular molecular entity.     

The substrate binding interface of alkylpurine DNA glycosylase AlkD

Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson–Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture.     

Structure-activity relationships of rationally designed AMACR 1A inhibitors

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the ‘racemisation’ reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure–activity relationship study has been performed. This paper describes the first structure–activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC₅₀ = 400–750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure–activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.     

Steroids from Ganoderma sinense as new natural inhibitors of cancer-associated mutant IDH1

Isocitrate dehydrogenase (IDH) is one of the key enzymes in the tricarboxylic acid cycle, and IDH mutations have been associated with many cancers, including glioblastoma, sarcoma, acute myeloid leukemia, etc. Three natural steroids 1–3 from Ganoderma sinense, a unique and rare edible-medicinal fungi in China, were found as potential IDH1 inhibitors by virtual ligand screening method. Among the three compounds, 3 showed the highest binding affinity to IDH1 with significant calculated binding free energy. Enzymatic kinetics demonstrated that 3 inhibited mutant enzyme in a noncompetitive manner. The half effective concentration of 3 for reducing the concentration of D-2HG in HT1080 cells was 35.97 μM. The levels of histone H3K9me3 methylation in HT1080 cells were reduced by treating with 3. Furthermore, knockdown of mutant IDH1 in HT1080 cells decreased the anti-proliferative sensitivity to 3. In short, our findings highlight that compound 3 may have clinical potential in tumor therapies as an effective inhibitor of mutant IDH1.     

Synthesis and biological evaluation of cis-restrained carbocyclic combretastatin A-4 analogs: Influence of the ring size and saturation on cytotoxic properties

Combretastatin A-4 (CA-4) is a highly cytotoxic natural product and several derivatives have been prepared which underwent clinical trial. These investigations revealed that the cis-stilbene moiety of the natural product is prone to undergo cis/trans isomerization under physiological conditions, reducing the overall activity of the drug candidates. Herein, we report the preparation of cis-restrained carbocyclic analogs of CA-4. The compounds, which differ by the size and hybridization of the carbocyclic ring have been evaluated for their cytotoxic properties and their ability to inhibit tubulin polymerization. Biological data, supported by molecular docking studies, identified cyclobutenyl and cyclobutyl derivatives of the natural product as highly promising drug candidates.     

Therapeutic utility of the newly discovered properties of interleukin-21

Since its discovery in 2000, interleukin-21 (IL-21) has been shown to display a broad spectrum of pleiotropic actions including the regulation of development, differentiation and function of lymphoid-myeloid cells. More specifically, IL-21 modulates the effector functions of T, B and NK cells, which not only have key roles in antitumoral and antiviral immunity but also in exerting major effects on inflammatory responses promoting the development of autoimmune diseases. Recent studies have unveiled an unexpected role for IL-21 in immune regulation and de novo T-cell development. While highlighting its critical role in immunity, this review will mainly focus on recent advances in IL-21 biology and how such newly discovered properties could potentially be exploited therapeutically in the establishment of future clinical trials.     

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F

ObjectivesBotulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored.MethodsIn present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model.ResultsThree compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (K_D: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC₅₀ of 30.47 ± 6.24, 14.91 ± 2.49 and 17.39 ± 2.74 μM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model.ConclusionNSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.     

Discovery of potent PTP1B inhibitors via structure-based drug design,synthesis and in vitro bioassay of Norathyriol derivatives

Protein tyrosine phosphatase 1B (PTP1B) has recently been identified as a potential target of Norathyriol. Unfortunately, Norathyriol is not a potent PTP1B inhibitor, which somewhat hinders its further application. Based on the fact that no study on the relationship of chemical structure and PTP1B inhibitory activity of Norathyriol has been reported so far, we attempted to perform structural optimization so as to improve the potency for PTP1B. Via structure-based drug design (SBDD), a rational strategy based on the binding mode of Norathyriol to PTP1B, we designed 26 derivatives with substitutions at the four phenolic hydroxyl groups of Norathyriol. By chemical synthesis and in vitro bioassay, we identified seven PTP1B inhibitors that were more potent than Norathyriol, of which XWJ24 showed the highest potency (IC₅₀: 0.6 μM). We also found out that XWJ24 was a competitive inhibitor and showed the 4.5-fold selectivity over its close homolog, TC-PTP. Through molecular docking of XWJ24 against PTP1B, we highlighted the essential role of its hydrogen bond with Asp181 for PTP1B inhibition and identified a potential halogen bond with Asp48 that was not observed for Norathyriol. The current data indicate that our SBDD strategy is effective to discover potent PTP1B-targeted Norathyriol derivatives, and XWJ24 is a promising lead compound for further development.     

Design,synthesis and evaluation of new classes of nonquaternary reactivators for acetylcholinesterase inhibited by organophosphates

A new series of nonquaternary conjugates for reactivation of both nerve agents and pesticides inhibited hAChE were described in this paper. It was found that substituted salicylaldehydes conjugated to aminobenzamide through piperidine would produce efficient reactivators for sarin, VX and tabun inhibited hAChE, such as L6M1R3, L6M1R5 to L6M1R7, L4M1R3 and L4M1R5 to L4M1R7. The in vitro reactivation experiment for pesticides inhibited hAChE of these new synthesized oximes were conducted for the first time. Despite they were less efficient than obidoxime, some of them were highlighted as equal or more efficient reactivators in comparison to 2-PAM. It was found that introduction of peripheral site ligands could increase oximes’ binding affinity for inhibited hAChE in most cases, which resulted in greater reactivation ability.     

Functional group and stereochemical requirements for substrate binding by ghrelin O-acyltransferase revealed by unnatural amino acid incorporation

Ghrelin is a small peptide hormone that undergoes a unique posttranslational modification, serine octanoylation, to play its physiological roles in processes including hunger signaling and glucose metabolism. Ghrelin O-acyltransferase (GOAT) catalyzes this posttranslational modification, which is essential for ghrelin to bind and activate its cognate GHS-R1a receptor. Inhibition of GOAT offers a potential avenue for modulating ghrelin signaling for therapeutic effect. Defining the molecular characteristics of ghrelin that lead to binding and recognition by GOAT will facilitate the development and optimization of GOAT inhibitors. We show that small peptide mimics of ghrelin substituted with 2,3-diaminopropanoic acid in place of the serine at the site of octanoylation act as submicromolar inhibitors of GOAT. Using these chemically modified analogs of desacyl ghrelin, we define key functional groups within the N-terminal sequence of ghrelin essential for binding to GOAT and determine GOAT’s tolerance to backbone methylations and altered amino acid stereochemistry within ghrelin. Our study provides a structure-activity analysis of ghrelin binding to GOAT that expands upon activity-based investigations of ghrelin recognition and establishes a new class of potent substrate-mimetic GOAT inhibitors for further investigation and therapeutic interventions targeting ghrelin signaling.     

Potential chemical transformation of phosphinic acid derivatives and their applications in the synthesis of drugs

The chemical transformation of phosphinic acid is a well-considered mature area of research on account of the historical significant reactions such as Kabachnik–Fields, Mannich, Arbuzov, Michaelis–Becker, etc. Considerable advances have been made over last years especially in metal-catalyzed, free-radical processes and asymmetric synthesis using catalytic enantioselective. As a result, the aim of this synopsis is to make the reader familiar with advances in the approaches of phosphinic acids toward the synthesis of highly functionalized and valuable buildings blocks. Another purpose of this survey is to provide the current status of the applications of phosphinic acids in the synthesis of drugs.     

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1^−/−Ifng^−/− mice with dimethylnitrosamine or carbon tetrachloride. Ifng^−/− and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1^−/−Ifng^−/− mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng^−/− mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.     

Bacillus thuringiensis toxin,Cry1C interacts with 128HLHFHLP134 region of aminopeptidase N of agricultural pest,Spodoptera litura

We modeled Cry1C toxin and its Aminopeptidase-N receptor and in silico docking analysis was performed. Further, we utilized biopanning against Cry1C followed by blocking assays and mutagenesis analysis to identify the binding epitope of SlAPN. We have identified a putative SlAPN binding region, APN-CRY (128HLHFHLP134). A derivative of SlAPN carrying the 128HLHFHLP134 region termed as binding region of APN (BR-APN) was cloned and its involvement in Cry1C binding and toxicity was checked. Cry1C-BR-APN binding was competed by synthetic peptides homologous to loop2 and loop3 of domain II but not by that of loopα. Additionally, alanines substitution of residues H128, H130, H132 and P134 affect the binding efficiency of receptor to Cry1C toxin (upto 4-fold lower affinity).These residues are also implicated in Cry1C toxicity as shown by the reduced ability to affect the mortality of Cry1C on S. litura larvae when toxin was preincubated with a fragment of the receptor.     

The adenosine A2A antagonistic properties of selected C8-substituted xanthines

The adenosine A_2A receptor is considered to be an important target for the development of new therapies for Parkinson’s disease. Several antagonists of the A_2A receptor have entered clinical trials for this purpose and many research groups have initiated programs to develop A_2A receptor antagonists. Most A_2A receptor antagonists belong to two different chemical classes, the xanthine derivatives and the amino-substituted heterocyclic compounds. In an attempt to discover high affinity A_2A receptor antagonists and to further explore the structure–activity relationships (SARs) of A_2A antagonism by the xanthine class of compounds, this study examines the A_2A antagonistic properties of series of (E)-8-styrylxanthines, 8-(phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines. The results document that among these series, the (E)-8-styrylxanthines have the highest binding affinities with the most potent homologue, (E)-1,3-diethyl-7-methyl-8-[(3-trifluoromethyl)styryl]xanthine, exhibiting a K_i value of 11.9 nM. This compound was also effective in reversing haloperidol-induced catalepsy in rats, providing evidence that it is in fact an A_2A receptor antagonist. The importance of substitution at C8 with the styryl moiety was demonstrated by the finding that none of the 8-(phenoxymethyl)xanthines and 8-(3-phenylpropyl)xanthines exhibited high binding affinities for the A_2A receptor.     

Synthesis,neuroprotective and antioxidant capacity of PBN-related indanonitrones

In this work six PBN-related indanonitrones 1–6 have been designed, synthesized, and their neuroprotection capacity tested in vitro, under OGD conditions, in SH-SY5Y human neuroblastoma cell cultures. As a result, we have identified indanonitrones 1, 3 and 4 (EC₅₀ = 6.64 ± 0.28 μM) as the most neuroprotective agents, and in particular, among them, indanonitrone 4 was also the most potent and balanced nitrone, showing antioxidant activity in three experiments [LOX (100 μM), APPH (51%), DPPH (36.5%)], being clearly more potent antioxidant agent than nitrone PBN. Consequently, we have identified (Z)-5-hydroxy-N-methyl-2,3-dihydro-1H-inden-1-imine oxide (4) as a hit-molecule for further investigation.     

Discovery of aromatic amides with triazole-core as potent reversal agents against P-glycoprotein-mediated multidrug resistance

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major impediment for clinical cancer therapy. 19 novel aromatic amides with triazole-core as MDR reversal agents were designed and synthesized via click chemistry to reverse MDR. Among them, compound 42 was identified as the most promising candidate with high potency (EC₅₀ = 78.1 ± 5.4 nM), low cytotoxity (SI > 1282) and persistent duration in reversing doxorubicin (DOX) resistance in K562/A02 cells. 42 also enhanced the potency of other P-gp associated cytotoxic agents with different structures. In further study, remarkably increased intracellular accumulation of Rh123 and DOX in K562/A02 cells was achieved by compound 42, while CYP3A4 activity had no change by compound 42. These results indicate that compound 42 as a relatively safe modulator of P-gp-mediated MDR has good potential for further development.