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.     

Single particle 3D reconstruction for 2D crystal images of membrane proteins

In cases where ultra-flat cryo-preparations of well-ordered two-dimensional (2D) crystals are available, electron crystallography is a powerful method for the determination of the high-resolution structures of membrane and soluble proteins. However, crystal unbending and Fourier-filtering methods in electron crystallography three-dimensional (3D) image processing are generally limited in their performance for 2D crystals that are badly ordered or non-flat. Here we present a single particle image processing approach, which is implemented as an extension of the 2D crystallographic pipeline realized in the 2dx software package, for the determination of high-resolution 3D structures of membrane proteins. The algorithm presented, addresses the low single-to-noise ratio (SNR) of 2D crystal images by exploiting neighborhood correlation between adjacent proteins in the 2D crystal. Compared with conventional single particle processing for randomly oriented particles, the computational costs are greatly reduced due to the crystal-induced limited search space, which allows a much finer search space compared to classical single particle processing. To reduce the considerable computational costs, our software features a hybrid parallelization scheme for multi-CPU clusters and computer with high-end graphic processing units (GPUs). We successfully apply the new refinement method to the structure of the potassium channel MloK1. The calculated 3D reconstruction shows more structural details and contains less noise than the map obtained by conventional Fourier-filtering based processing of the same 2D crystal images.     

Activin signalling and pre-eclampsia: From genetic risk to pre-symptomatic biomarker

Pre-eclampsia is a multi-system condition in pregnancy that is characterised by the onset of hypertension and proteinuria in women after the 20th week and it remains a leading cause of maternal and fetal mortality. Despite this the causative molecular basis of pre-eclampsia remains poorly understood. As a result, an intensive research effort has focused on understanding the molecular mechanisms involved in pre-eclampsia and using this information to identify new pre-symptomatic bio-markers of the condition. Activin A and its receptor, ACVR2A, have been extensively studied in this regard.Activin A is a member of the transforming growth factor (TGF)-β superfamily that has a wide range of biological functions depending on the cellular context. Recent work has shown that polymorphisms in ACVR2A may be a genetic risk factor for pre-eclampsia. Furthermore, both placenta and serum levels of Activin A are significantly increased in pre-eclampsia suggesting that Activin A may be a possible biomarker for the condition. Here we review the latest advances in this field and link these with new molecular data that suggest that the oxidative stress and pro-inflammatory cytokine production seen in pre-eclampsia may result in increased placental Activin A secretion in an attempt to maintain placental function.     

C5a receptor-targeting ligand-mediated delivery of dengue virus antigen to M cells evokes antigen-specific systemic and mucosal immune responses in oral immunization

Oral mucosal immunization is a feasible and economic vaccination strategy. In order to achieve a successful oral mucosal vaccination, antigen delivery to gut immune inductive site and avoidance of oral tolerance induction should be secured. One promising approach is exploring the specific molecules expressed on the apical surfaces of M cells that have potential for antigen uptake and immune stimulation. We previously identified complement 5a receptor (C5aR) expression on human M-like cells and mouse M cells and confirmed its non-redundant role as a target receptor for antigen delivery to M cells using a model antigen. Here, we applied the OmpH ligand, which is capable of targeting the ligand-conjugated antigen to M cells to induce specific mucosal and systemic immunities against the EDIII of dengue virus (DENV). Oral immunization with the EDIII–OmpH efficiently targeted the EDIII to M cells and induced EDIII-specific immune responses comparable to those induced by co-administration of EDIII with cholera toxin (CT). Also, the enhanced responses by OmpH were characterized as Th2-skewed responses. Moreover, oral immunization using EDIII–OmpH did not induce systemic tolerance against EDIII. Collectively, we suggest that OmpH-mediated targeting of antigens to M cells could be used for an efficient oral vaccination against DENV infection.     

The LC8-RavP ensemble Structure Evinces A Role for LC8 in Regulating Lyssavirus Polymerase Functionality

The rabies and Ebola viruses recruit the highly conserved host protein LC8 for their own reproductive success. In vivo knockouts of the LC8 recognition motif within the rabies virus phosphoprotein (RavP) result in completely nonlethal viral infections. In this work, we examine the molecular role LC8 plays in viral lethality. We show that RavP and LC8 colocalize in rabies infected cells, and that LC8 interactions are essential for efficient viral polymerase functionality. NMR, SAXS, and molecular modeling demonstrate that LC8 binding to a disordered linker adjacent to an endogenous dimerization domain results in restrictions in RavP domain orientations. The resulting ensemble structure of RavP-LC8 tetrameric complex is similar to that of a related virus phosphoprotein that does not bind LC8, suggesting that with RavP, LC8 binding acts as a switch to induce a more active conformation. The high conservation of the LC8 motif in Lyssavirus phosphoproteins and its presence in other analogous proteins such as the Ebola virus VP35 evinces a broader purpose for LC8 in regulating downstream phosphoprotein functions vital for viral replication.     

Promising anti-inflammatory effects of chalcones via inhibition of cyclooxygenase,prostaglandin E2, inducible NO synthase and nuclear factor κb activities

Chalcones (1, 3-Diphenyl-2-propen-1-one) consist of a three carbon α, β-unsaturated carbonyl system and act as precursors for the biosynthesis of flavonoids in plants. However, laboratory synthesis of various chalcones has also been reported. Both natural and synthetic chalcones are known to exhibit a variety of pharmacological activities such as anti-inflammatory, antitumor, antibacterial, antifungal, antimalarial and antituberculosis. These promising activities, ease of synthesis and simple chemical structure have awarded chalcones considerable attraction. This review focuses on the anti-inflammatory effects of chalcones, caused by their inhibitory action primarily against the activities and expressions of four key inflammatory mediators viz., cyclooxygenase, prostaglandin E₂, inducible NO synthase, and nuclear factor κB. Various methodologies for the synthesis of chalcones have been discussed. The potency of recently synthesized chalcones is given in terms of their IC₅₀ values. Structure-Activity Relationships (SARs) of a variety of chalcone derivatives have been discussed. Computational methods were applied to calculate the ideal orientation of a typical chalcone scaffold against three enzymes, namely, cyclooxygenase-1, cyclooxygenase-2 and inducible NO synthase for the formation of stable complexes. The global market of anti-inflammatory drugs and its expected growth (from 2018 to 2026) have been discussed. SAR analysis, docking studies, and future prospects all together provide useful clues for the synthesis of novel chalcones of improved anti-inflammatory activities.     

Proteome variation of the rat liver after static cold storage assayed in an ex vivo model

Cold storage is a common procedure for liver preservation in a transplant setting. However, during cold ischemia, the liver suffers molecular alterations that can affect its performance. Also, deleterious mechanisms set forth in the storage phase are exacerbated during reperfusion. This study aimed to identify liver proteins associated with injury during cold storage and/or normothermic reperfusion using the isolated perfused rat liver model. Livers from male rats were subjected to either (1) cold storage for 24 h, (2) ex vivo normothermic reperfusion for 90 min or (3) cold storage for 24 h followed by ex vivo normothermic reperfusion for 90 min. Then, the livers were homogenized and proteins were extracted. Protein expression between each experimental group and the control (freshly resected livers) was compared by two-dimensional (2D) gel electrophoresis. Protein identification was carried out by matrix‐assisted laser desorption/ionization time‐of‐flight spectrometry (MALDI‐TOF/TOF) using MASCOT as the search engine. 23 proteins were detected with significantly altered levels of expression among the different treatments, including molecular chaperones, antioxidant enzymes, and proteins involved in energy metabolism. Some of them have been postulated as biomarkers for liver damage while others had been identified in other organs subjected to ischemia and reperfusion injury. The whole data set will be a useful resource for studying deleterious molecular mechanisms that result in diminished liver function during storage and subsequent reperfusion.     

Discovery of amide-bridged pyrrolo[2,3-d]pyrimidines as tumor targeted classical antifolates with selective uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis

We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) α and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FRα and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1 kcal/mol. Compound 7 showed ~3-fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FRα over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FRα-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a “first-in-class” classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FRα over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.     

Chemically induced degradation of CK2 by proteolysis targeting chimeras based on a ubiquitin–proteasome pathway

As a ubiquitous, highly pleiotropic and constitutively active serine/threonine protein kinase, casein kinase 2 (CK2) is closely associated with tumorigenesis by its overexpression in cancer cells. Here we report several proteolysis targeting chimeras (PROTACs) via “click reaction” to connect a CK2 inhibitor (CX-4945) and pomalidomide for degradation of CK2 protein. Among them, compound 2 degraded CK2 in a dose and time-dependent manner, and kept CK2 at a low basal level by recruiting ubiquitin-proteasome system. The degradation of CK2 resulted in the reduced phosphorylation of Akt and the up-regulation of p53. As a CK2 protein degrader, 2 showed the analogous cytotoxicity to CX-4945 but with a quite different mechanism of action from the CK2 inhibitor, hinting that degradation of CK2 proteins by PROTACs is a potential way for cancer treatments.     

X-ray Structures of the Post-fusion 6-Helix Bundle of the Human Syncytins and their Functional Implications

The retroviral envelope-derived proteins syncytin-1 and syncytin-2 (syn1 and syn2) drive placentation in humans by forming a syncytiotophoblast, a structure allowing for an exchange interface between maternal and fetal blood during pregnancy. Despite their essential role, little is known about the molecular mechanism underlying the syncytins' function. We report here the X-ray structures of the syn1 and syn2 transmembrane subunit ectodomains, featuring a 6-helix bundle (6HB) typical of the post-fusion state of gamma-retrovirus and filovirus fusion proteins. Contrary to the filoviruses, for which the fusion glycoprotein was crystallized both in the post-fusion and in the spring-loaded pre-fusion form, the highly unstable nature of the syncytins' prefusion form has precluded structural studies. We undertook a proline-scanning approach searching for regions in the syn1 6HB central helix that tolerate the introduction of helix-breaker residues and still fold correctly in the pre-fusion form. We found that there is indeed such a region, located two α-helical turns downstream a stutter in the central coiled-coil helix - precisely where the breaks of the spring-loaded helix of the filoviruses map. These mutants were fusion-inactive as they cannot form the 6HB, similar to the “SOSIP” mutant of HIV Env that allowed the high-resolution structural characterization of its labile pre-fusion form. These results now open a new window of opportunity to engineer more stable variants of the elusive pre-fusion trimer of the syncytins and other gamma-retroviruses envelope proteins for structural characterization.     

Rationally designed molecules for resurgence of cyanide mitigated cytochrome c oxidase activity

Cytochrome c oxidase (CcOX) containing binuclear heme a₃-Cu B centre (BNC) mechanises the process of electron transfer in the last phase of cellular respiration. The molecular modelling based structural analysis of CcOX – heme a₃-Cu B complex was performed and the disturbance to this complex under cyanide poisoning conditions was investigated. Taking into consideration the results of molecular docking studies, new chemical entities were developed for clipping cyanide from the enzyme and restoring its normal function. It was found that the molecules obtained by combining syringaldehyde, oxindole and chrysin moieties bearing propyl/butyl spacing groups occupy the BNC region and effectively remove cyanide bound to the enzyme. The binding constant of compound 2 with CN^– was 2.3 × 10⁵ M⁻¹ and its ED₅₀ for restoring the cyanide bound CcOX activity in 10 min was 16 µM. The compound interacted with CN^– over the pH range 5–10. The comparison of the loss of enzymatic activity in the presence of CN^– and resumption of enzymatic activity by compound 2 mediated removal of CN^– indicated the efficacy of the compound as antidote of cyanide.     

Impact of the natural resource of UVB on the content of vitamin D2 in oyster mushroom (Pleurotus ostreatus) under subtropical settings

Vitamin D deficiency is a pandemic problem. Non-animal source of vitamin D is obtained from edible mushrooms. Oyster mushroom (Pleurotus ostreatus) was sliced into the size of 1 cm³, 4 cm³ and 9 cm³, and treated with the sun as a natural resource of UVB under subtropical settings in Ethiopia. The content of vitamin D was measured by using high-performance liquid chromatography (HPLC). After sun treatment, there was a significant increment in the content of vitamin D₂ from nil to 67.4 ± 28.0 µg/g dry weight (DW). Based on the results of the overall pairwise comparisons, 1 cm³ size of slice group had the highest content of vitamin D₂. Duration of sun exposure, sizes of mushroom slices and moisture content were identified as determining factors for vitamin D₂ synthesis. Exposing slices of oyster mushroom to the sunlight for <30 min provides the amount that satisfies the current recommended dietary allowance (RDA) of vitamin D without any visible change in color and texture. Thus, sun treatment of oyster mushroom is an effective and economically cheap strategy in the fight against vitamin D deficiency.     

Lambertellin from Pycnoporus sanguineus MUCL 51321 and its anti-inflammatory effect via modulation of MAPK and NF-κB signaling pathways

Lambertellin (1) and ergosta-5,7,22-trien-3-ol (2) were isolated from the solid rice fermentation of the plant pathogenic fungus Pycnoporus sanguineus MUCL 51321. Their structures were elucidated using comprehensive spectroscopic methods. The isolated compounds were tested on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Lambertellin (1) exhibited promising inhibitory activity against nitric oxide (NO) production with IC₅₀ value of 3.19 µM, and it significantly inhibited the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). Lambertellin (1) also decreased the expression of pro-inflammatory cytokines IL-6 and IL-1β. The study of the mechanistic pathways revealed that lambertellin (1) exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophage cells by modulating the activation of the mitogen activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways. Therefore, lambertellin (1) could be a promising lead compound for the development of new anti-inflammatory drugs.     

Cytotoxic mechanism of novel compound jiangxienone from Cordyceps jiangxiensis against cancer cells involving DNA damage response pathway

Jiangxienone is a novel compound recently purified from the traditional Chinese medicinal mushroom Cordyceps jiangxiensis and was reported to show potent cytotoxicity against cancer cells. However, its mechanism of action remains unclear. In this work, the underlying mechanism of jiangxienone against human gastric cancer cells HGC-27 was investigated using whole-genome microarray. The results demonstrated that jiangxienone significantly decreased cell population of various human cancer cell lines, while slightly inhibited the colony formation of stromal cells from murine marrow even at a high concentration. Differential gene expression profiling indicated that the cytotoxic action of jiangxienone against HGC-27 is closely related to the DNA damage response pathway, which was evident by the identification of 23 DNA damage response-associated genes, such as XRCC4/5/6, NBS1, RAD51, and BRCA1/2. By using gel retardation assays, UV absorption spectrometry and single-cell gel electrophoresis, it was found that jiangxienone could bind to DNA and inhibit cancer cell growth. The above results indicated that the cytotoxic mechanism of jiangxienone against cancer cells was involved in the DNA damage response pathway. The findings will be helpful to the development of useful cancer chemopreventive compounds from C. jiangxiensis.     

Novel FXR (farnesoid X receptor) modulators: Potential therapies for cholesterol gallstone disease

Metabolic disorders such as diabetes are known risk factors for developing cholesterol gallstone disease (CGD). Cholesterol gallstone disease is one of the most prevalent digestive diseases, leading to considerable financial and social burden worldwide. Ursodeoxycholic acid (UDCA) is the only bile acid drug approved by FDA for the non-surgical treatment of gallstones. However, the molecular link between UDCA and CGD is unclear. Previous data suggest that the farnesoid X receptor (FXR), a bile acid nuclear receptor, may protect against the development of CGD. In studies aimed at identifying the role of FXR, we recently identify a novel chemical tool, 6EUDCA (6-αethyl-ursodeoxycholic acid), a synthetic derivative of UDCA, for studying FXR. We found that 6EUDCA binds FXR stronger than UDCA in a TR-FRET binding assay. This result was supported by computational docking models that suggest 6EUDCA forms a more extensive hydrogen bound network with FXR. Interestingly, neither compound could activate FXR target genes in human nor mouse liver cells, suggesting UDCA and 6EUDCA activate non-genomic signals in an FXR-dependent manner. Overall these studies may lead to the identification of a novel mechanism by which bile acids regulate cell function, and 6EUDCA may be an effective targeted CGD therapeutic.     

Synthesis and evaluation of an AZD2461 [18F]PET probe in non-human primates reveals the PARP-1 inhibitor to be non-blood-brain barrier penetrant

Poly(ADP-ribose)polymerase-1 inhibitor (PARPi) AZD2461 was designed to be a weak P-glycoprotein (P-gp) analogue of FDA approved olaparib. With this chemical property in mind, we utilized the AZD2461 ligand architecture to develop a CNS penetrant and PARP-1 selective imaging probe, in order to investigate PARP-1 mediated neuroinflammation and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Our work led to the identification of several high-affinity PARPi, including AZD2461 congener 9e (PARP-1 IC₅₀ = 3.9 ± 1.2 nM), which was further evaluated as a potential ¹⁸F-PET brain imaging probe. However, despite the similar molecular scaffolds of 9e and AZD2461, our studies revealed non-appreciable brain-uptake of [¹⁸F]9e in non-human primates, suggesting AZD2461 to be non-CNS penetrant.     

The development of 2-acetylphenol-donepezil hybrids as multifunctional agents for the treatment of Alzheimer’s disease

A series of 2-acetylphenol-donepezil hybrids was designed and synthesized based on multi-target-directed ligands strategy. The biological activities were evaluated by AChE/BChE inhibition and MAO-A/MAO-B inhibition. The results revealed that the tertiary amines and methylene chain length significantly affected the eeAChE inhibitory potency, in particular, compound TM-14 showed the best eeAChE inhibitory activity with IC₅₀ value of 2.9 μM, in addition, both kinetic analysis of AChE inhibition and docking study displayed that TM-14 could simultaneously bind to the catalytic active site and peripheral anionic site of AChE. Moreover, compound TM-14 was a selective metal chelator and could form 1:1 TM-14-Cu²⁺ complex. The structure-active-relationship also indicated that the O-alkylamine fragment remarkably decreased hMAO-B inhibitory activity, compound TM-2 exhibited potent hMAO-B inhibitory activity (IC₅₀ = 6.8 μM), which was supported by the molecular docking study. More interestingly, compounds TM-14 and TM-2 could cross the blood-brain barrier in vitro. Therefore, the structure-active-relationship of 2-acetylphenol-donepezil hybrids could encourage the development of multifunction agents with selective AChE inhibition or selective MAO-B inhibition for the treatment of Alzheimer’s disease.     

Is the Sudlow site I of human serum albumin more generous to adopt prospective anti-cancer bioorganic compound than that of bovine: A combined spectroscopic and docking simulation approach

A comparative biophysical study on the individual conformational adaptation embraced by two homologous serum albumins (SA) (bovine and human) towards a potential anticancer bioorganic compound 2-(6-chlorobenzo[d] thiazol-2-yl)-1H-benzo[de] isoquinoline-1,3(2H)- dione (CBIQD) is apparent from the discrimination in binding behavior and the ensuing consequences accomplished by combined in vitro optical spectroscopy, in silico molecular docking and molecular dynamics (MD) simulation. The Sudlow site I of HSA although anion receptive, harbors neutral CBIQD in Sudlow site I (subdomain IIA, close to Trp) of HSA, while in BSA its prefers to snugly fit into Sudlow site II (subdomain IIIA, close to Tyr). Based on discernable diminution of HSA mean fluorescence lifetime as a function of biluminophore concentration, facile occurrence of fluorescence resonance energy transfer (FRET) is substantiated as the probable quenching mechanism accompanied by structural deformations in the protein ensemble. CBIQD establishes itself within HSA close to Trp214, and consequently reduces the micropolarity of the cybotactic environment that is predominantly constituted by hydrophobic amino acid residues. The stronger association of CBIQD with HSA encourages an allosteric modulation leading to slight deformation in its secondary structure whereas for BSA the association is comparatively weaker. Sudlow site I of HSA is capable to embrace a favorable conformation like malleable gold to provide room for incoming CBIQD, whereas for BSA it behaves more like rigid cast-iron which does not admit any change thus forcing CBIQD to occupy an altogether different binding location i.e. the Sudlow site II. The anticancer CBIQD is found to be stable within the HSA scaffold as vindicated by root mean square deviation (RMSD) and root mean square fluctuation (RMSF) obtained by MD simulation. A competitively inhibited esterase-like activity of HSA upon CBIQD binding to Lys199 and Arg257 residues, plausibly envisions that similar naphthalimide based prodrugs, bearing ester functionality, can be particularly activated by Sudlow site I of HSA. The consolidated spectroscopic research described herein may encourage design of naphthalimide based pro-drugs for effective in vivo biodistribution using HSA-based drug delivery systems.