Advances and prospects of analytic methods for bacterial transglycosylation and inhibitor discovery.

The cell wall is essential for bacteria to maintain structural rigidity and withstand external osmotic pressure. In bacteria, the cell wall is composed of peptidoglycan. Lipid II is the basic unit for constructing highly cross-linked peptidoglycan scaffolds. Transglycosylase (TGase) is the initiating enzyme in peptidoglycan synthesis that catalyzes the ligation of lipid II moieties into repeating GlcNAc-MurNAc polysaccharides, followed by transpeptidation to generate cross-linked structures. In addition to the transglycosylases in the class-A penicillin-binding proteins (aPBPs), SEDS (shape, elongation, division and sporulation) proteins are also present in most bacteria and play vital roles in cell wall renewal, elongation, and division. In this review, we focus on the latest analytical methods including the use of radioactive labeling, gel electrophoresis, mass spectrometry, fluorescence labeling, probing undecaprenyl pyrophosphate, fluorescence anisotropy, ligand-binding-induced tryptophan fluorescence quenching, and surface plasmon resonance to evaluate TGase activity in cell wall formation. This review also covers the discovery of TGase inhibitors as potential antibacterial agents. We hope that this review will give readers a better understanding of the chemistry and basic research for the development of novel antibiotics.

Degradation of neurodegenerative disease-associated TDP-43 aggregates and oligomers via a proteolysis-targeting chimera.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43. METHODS: By applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43. RESULTS: Four PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system. CONCLUSIONS: Our study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.

Revisiting Disulfide-Yne and Disulfide-Diazonium Reactions for Potential Direct Modification of Disulfide Bonds in Proteins.

To find their potential use in protein research, direct addition of a disulfide compound to alkyne (namely disulfide-yne reaction) and S-arylation with arenediazonium salt (namely disulfide-diazonium reaction) were investigated in aqueous or protic solutions. The reaction of dimethyl disulfide with 5-hexynol performed best under 300 nm irradiation in the presence of sodium acetate to afford 5,6-bis(methylthio)-5-hexenol in 60% yield. Without the prior reduction of a disulfide bond to thiols, the disulfide-yne reactions have the advantage of 100% atom economy. Disulfide-diazonium reaction was triggered by sodium formate and accelerated by photoirradiation with a 450 nm LED lamp (5 W). The reaction of 3,4-dihydroxy-1,2-dithiane with 2-(prop-2-yn-1-yloxy)benzene-1-diazonium tetrafluoroborate (8b) afforded 2-(benzofuran-3-yl)-1,3-dithiepane-5,6-diol (13), confirming that both S substituents originate from the same disulfide molecule. The trastuzumab antibody was incubated with diazonium 8b, followed by α-lytic protease digestion, LC-ESI-MS/MS analysis, and Mascot search, to verify that the proximal C229 and C232 residues on the same heavy chain were reconnected with a (benzofuranyl)methine moiety that originated from 8b, unlike the expected disulfide rebridging across two heavy chains. Nonetheless, disulfide-diazonium reactions still have potential for rebridging disulfide bonds if appropriate proteins and diazonium agents are chosen.

Photoaffinity labeling of benzophenone-containing salicylanilide compounds to give an insight into the mechanism in disrupting peptidoglycan formation.

A series of salicylanilide compounds was previously identified as antibacterial agents that inhibit the peptidoglycan formation. To find the exact binding mode, we synthesized a benzophenone-containing salicylanilide compound (1) and used it as a photoaffinity probe to label Acinetobacter baumannii penicillin-binding protein (PBP1b). After incubation and photo-irradiation, the labeled protein was subjected to trypsin digestion, dialysis enrichment, LC-ESI-MS/MS analysis, and Mascot search to reveal an octadecapeptide sequence 364RQLRTEYQESDLTNQGLR381 that was labeled at E372. Our molecular docking experiments suggest a hydrophobic pocket surrounded by R367 and E372 is the binding site of salicylanilide 1. The pocket lies in between the transglycosylase and transpeptidase domains, thus binding of salicylanilide 1 can block the propagation pathway to disrupt the growth of peptidoglycan chain.

Effective assay of bacterial transglycosylation by molecular turn-on sensing and a second-order scattering effect.

Instead of using the lipid II substrate that requires prior labelling with a radioactive isotope or fluorophore to probe the formation of peptidoglycan in bacterial transglycosylation, the released undecaprenyl pyrophosphate (UPP) product is quantitatively measured either using a terpyridine-zinc fluorescence turn-on sensor or simply by the second-order scattering effect of the in situ formed UPP-calcium complex. Both the assay methods are utilized to identify moenomycin A as a potent transglycosylase inhibitor with a consistent IC50 value.

The Ca-loop in thymidylate kinase is critical for growth and contributes to pyrimidine drug sensitivity of Candida albicans.

The yeast Candida albicans is the most prevalent opportunistic fungal pathogen in humans. Drug resistance among C. albicans isolates poses a common challenge, and overcoming this resistance represents an unmet need in managing this common pathogen. Here, we investigated CDC8, encoding thymidylate kinase (TMPK), as a potential drug target for the management of C. albicans infections. We found that the region spanning amino acids 106-123, namely the Ca-loop of C. albicans TMPK (CaTMPK), contributes to the hyperactivity of this enzyme compared with the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/deoxy-5-fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, expression of CaTMPK, but not of hTMPK, produced dUTP/5-FdUTP-mediated DNA toxicity in budding yeast (Saccharomyces cerevisiae). CRISPR-mediated deletion of this Ca-loop in C. albicans revealed that the Ca-loop is critical for fungal growth and susceptibility to 5-fluorouridine (5-FUrd). Of note, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd, and we also found that CaTMPK is essential for the growth of C. albicans In conclusion, these findings not only identified a target site for the development of CaTMPK-selective drugs, but also revealed that 5-FUrd may have potential utility as drug for managing C. albicans infections.

Constructing conjugate vaccine against Salmonella Typhimurium using lipid-A free lipopolysaccharide.

BACKGROUND: Salmonella enterica serotype Typhimurium is a nontyphoidal and common foodborne pathogen that causes serious threat to humans. There is no licensed vaccine to prevent the nontyphoid bacterial infection caused by S. Typhimurium. METHODS: To develop conjugate vaccines, the bacterial lipid-A free lipopolysaccharide (LFPS) is prepared as the immunogen and used to synthesize the LFPS-linker-protein conjugates 6a-9b. The designed bifunctional linkers 1-5 comprising either an o-phenylenediamine or amine moiety are specifically attached to the exposed 3-deoxy-D-manno-octulosonic acid (Kdo), an α-ketoacid saccharide of LFPS, via condensation reaction or decarboxylative amidation. In addition to bovine serum albumin and ovalbumin, the S. Typhimurium flagellin (FliC) is also used as a self-adjuvanting protein carrier. RESULTS: The synthesized conjugate vaccines are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC), and their contents of polysaccharides and protein are determined by phenol-sulfuric acid assay and bicinchoninic acid assay, respectively. Enzyme-linked immunosorbent assay (ELISA) shows that immunization of mouse with the LFPS-linker-protein vaccines at a dosage of 2.5 µg is sufficient to elicit serum immunoglobulin G (IgG) specific to S. Typhimurium lipopolysaccharide (LPS). The straight-chain amide linkers in conjugates 7a-9b do not interfere with the desired immune response. Vaccines 7a and 7b derived from either unfractionated LFPS or the high-mass portion show equal efficacy in induction of IgG antibodies. The challenge experiments are performed by oral gavage of S. Typhimurium pathogen, and vaccine 7c having FliC as the self-adjuvanting protein carrier exhibits a high vaccine efficacy of 74% with 80% mice survival rate at day 28 post the pathogen challenge. CONCLUSIONS: This study demonstrates that lipid-A free lipopolysaccharide prepared from Gram-negative bacteria is an appropriate immunogen, in which the exposed Kdo is connected to bifunctional linkers to form conjugate vaccines. The decarboxylative amidation of Kdo is a novel and useful method to construct a relatively robust and low immunogenic straight-chain amide linkage. The vaccine efficacy is enhanced by using bacterial flagellin as the self-adjuvanting carrier protein.

A Terpyridine Zinc Complex for Selective Detection of Lipid Pyrophosphates: A Model System for Monitoring Bacterial O- and N-Transglycosylations.

To develop an effective method for probing O- and N-glycosyltransfer reactions that are accompanied by the release of undecaprenyl pyrophosphate, solanesyl pyrophosphate (SPP) is used as a surrogate to bind a terpyridine zinc complex (Tpy-Zn), forming a fluorescent [Tpy-Zn]-SPP complex (Kass 106,000 M-1 in EtOH-CHCl3) with 5.8 µM LOD in HEPES buffer (10 mM, pH 7.4) containing 10 mM CaCl2 and 0.08% decyl PEG, which is similar to the bioassay conditions for lipid II polymerization.

Development of effective anti-influenza drugs: congeners and conjugates - a review.

Influenza is a long-standing health problem. For treatment of seasonal flu and possible pandemic infections, there is a need to develop new anti-influenza drugs that have good bioavailability against a broad spectrum of influenza viruses, including the resistant strains. Relenza™ (zanamivir), Tamiflu™ (the phosphate salt of oseltamivir), Inavir™ (laninamivir octanoate) and Rapivab™ (peramivir) are four anti-influenza drugs targeting the viral neuraminidases (NAs). However, some problems of these drugs should be resolved, such as oral availability, drug resistance and the induced cytokine storm. Two possible strategies have been applied to tackle these problems by devising congeners and conjugates. In this review, congeners are the related compounds having comparable chemical structures and biological functions, whereas conjugate refers to a compound having two bioactive entities joined by a covalent bond. The rational design of NA inhibitors is based on the mechanism of the enzymatic hydrolysis of the sialic acid (Neu5Ac)-terminated glycoprotein. To improve binding affinity and lipophilicity of the existing NA inhibitors, several methods are utilized, including conversion of carboxylic acid to ester prodrug, conversion of guanidine to acylguanidine, substitution of carboxylic acid with bioisostere, and modification of glycerol side chain. Alternatively, conjugating NA inhibitors with other therapeutic entity provides a synergistic anti-influenza activity; for example, to kill the existing viruses and suppress the cytokines caused by cross-species infection.

Site-Selective Functionalization of Flagellin by Steric Self-Protection: A Strategy To Facilitate Flagellin as a Self-Adjuvanting Carrier in Conjugate Vaccine.

Flagellin (FliC) can act as a carrier protein in the preparation of conjugate vaccines to elicit a T-cell-dependent immune response and as an intrinsic adjuvant to activate the toll-like receptor 5 (TLR5) to enhance vaccine potency. To enable the use of FliC as a self-adjuvanting carrier, an effective method for site-selective modification (SSM) of pertinent amino-acid residues in the D2 and D3 domains of FliC is explored without excessive modification of the D0 and D1 domains, which are responsible for activating and binding with TLR5. In highly concentrated Na2 SO4 solution, FliC monomers form flagellar filaments, in which the D0 and D1 domains are situated inside the tubular structure. Thus, the lysine residues (K219, K224, K324, and K331) in the D2 and D3 domains of flagellin are selectively modified by a diazo-transfer reaction with imidazole-1-sulfonyl azide. The sites with azido modification are confirmed by MALDI-TOF-MS, ESI-TOF-MS, and LC-MS/MS analyses along with label-free quantitation. The azido-modified filament dissolves to give FliC monomers, which can conjugate with alkyne-hinged saccharides by the click reaction. Transmission electron microscopy imaging, dynamic light scattering measurements, and the secreted embryonic alkaline phosphatase reporter assay indicate that the modified FliC monomers retain the ability either to bind with TLR5 or to reassemble into filaments. Overall, this study establishes a feasible method for the SSM of FliC by steric self-protection of the D0 and D1 domains.

Activation of AMP-activated protein kinase α1 mediates mislocalization of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein-43 (TDP-43) is a nuclear RNA-binding protein involved in many cellular pathways. TDP-43-positive inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). The major clinical presentation of ALS is muscle weakness due to the degeneration of motor neurons. Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early event of ALS. In this study, we demonstrate that cytoplasmic mislocalization of TDP-43 was accompanied by increased activation of AMP-activated protein kinase (AMPK) in motor neurons of ALS patients. The activation of AMPK in a motor neuron cell line (NSC34) or mouse spinal cords induced the mislocalization of TDP-43, recapitulating this characteristic of ALS. Down-regulation of AMPK-α1 or exogenous expression of a dominant-negative AMPK-α1 mutant reduced TDP-43 mislocalization. Suppression of AMPK activity using cAMP-simulating agents rescued the mislocalization of TDP-43 in NSC34 cells and delayed disease progression in TDP-43 transgenic mice. Our findings demonstrate that activation of AMPK-α1 plays a critical role in TDP-43 mislocalization and the development of ALS; thus, AMPK-α1 may be a potential drug target for this devastating disease.

Peramivir analogues bearing hydrophilic side chains exhibit higher activities against H275Y mutant than wild-type influenza virus.

Peramivir is an effective anti-influenza drug in the clinical treatment of influenza, but its efficacy toward the H275Y mutant is reduced. The previously reported cocrystal structures of inhibitors in the mutant neuraminidase (NA) suggest that the hydrophobic side chain should be at the origin of reduced binding affinity. In contrast, zanamivir having a hydrophilic glycerol side chain still possesses high affinity toward the H275Y NA. We thus designed five peramivir analogues (5-9) carrying hydrophilic glycol or glycerol side chains, and evaluated their roles in anti-influenza activity, especially for the H275Y mutant. The synthetic sequence involves a key step of (3 + 2) cycloaddition reactions between alkenes and nitrile oxides to construct the scaffold of peramivir carrying the desired hydrophilic side chains and other appropriate functional groups. The molecular docking experiments reveal that the hydrophilic side chain can provide extra hydrogen bonding with the translocated Glu-276 residue in the H275Y NA active site. Thus, the H275Y mutant may be even more sensitive than wild-type virus toward the peramivir analogues bearing hydrophilic side chains. Notably, the peramivir analogue bearing a glycerol side chain inhibits the H275Y mutant with an IC50 value of 35 nM, which is better than the WSN virus by 9 fold.

Cell-permeable probe for identification and imaging of sialidases.

Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl-enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.

Tracking and Finding Slow-Proliferating/Quiescent Cancer Stem Cells with Fluorescent Nanodiamonds.

Quiescent cancer stem cells (CSCs) have long been considered to be a source of tumor initiation. However, identification and isolation of these cells have been hampered by the fact that commonly used fluorescent markers are not sufficiently stable, both chemically and photophysically, to allow tracking over an extended period of time. Here, it is shown that fluorescent nanodiamonds (FNDs) are well suited for this application. Genotoxicity tests of FNDs with comet and micronucleus assays for human fibroblasts and breast cancer cells indicate that the nanoparticles neither cause DNA damage nor impair cell growth. Using AS-B145-1R breast cancer cells as the model cell line for CSC, it is found that the FND labeling outperforms 5-ethynyl-2'-deoxyuridine (EdU) and carboxyfluorescein diacetate succinimidyl ester (CFSE) in regards to its long-term tracking capability (>20 d). Moreover, through a quantification of their stem cell activity by measuring mammosphere-forming efficiencies (MFEs) and self-renewal rates, the FND-positive cells are identified to have an MFE twice as high as that of the FND-negative cells isolated from the same dissociated mammospheres. Thus, the nanoparticle-based labeling technique provides an effective new tool for tracking and finding slow-proliferating/quiescent CSCs in cancer research.

A short synthesis of (±)-antroquinonol in an unusual scaffold of 4-hydroxy-2-cyclohexenone.

Antroquinonol, which was first isolated from a mushroom, Antrodia cinnamomea, found in Taiwan, is an anticancer compound with a unique core structure of 4-hydroxy-2,3-dimethoxycyclohex-2-enone carrying methyl, farnesyl and hydroxyl substituents in the 4,5-cis-5,6-trans configuration. A short synthesis of (±)-antroquinonol is accomplished in seven steps from 2,3,4-trimethoxyphenol, which is oxidized in methanol to a highly electron-rich substrate of 2,3,4,4-tetramethoxycyclohexadienone and then a Michael reaction with dimethylcuprate is performed as the key step, followed by alkylation, reduction and epimerization to incorporate the required substituents at three contiguous stereocenters.

An azido-BODIPY probe for glycosylation: initiation of strong fluorescence upon triazole formation.

We have designed a low fluorescent azido-BODIPY-based probe AzBOCEt (Az10) that undergoes copper(I)-catalyzed 1,3-dipolar cycloadditions with alkynes to yield strongly fluorescent triazole derivatives. The fluorescent quantum yield of a triazole product T10 is enhanced by 52-fold as compared to AzBOCEt upon excitation at a wavelength above 500 nm. Quantum mechanical calculations indicate that the increase in fluorescence upon triazole formation is due to the lowering of the HOMO energy level of the aryl moiety to reduce the process of acceptor photoinduced electron transfer. AzBOCEt is shown to label alkyne-functionalized proteins in vitro and glycoproteins in cells with excellent selectivity, and enables cell imaging and visualization of glycoconjugates in alkynyl-saccharide-treated cells at extremely low concentration (0.1 µM). Furthermore, the alkyne-tagged glycoproteins from cell lysates can be directly detected with AzBOCEt in gel electrophoresis.

Chemical constituents of Plectranthus amboinicus and the synthetic analogs possessing anti-inflammatory activity.

This study demonstrates that compounds 1-4 from an extract of Plectranthus amboinicus inhibit the binding of AP-1 to its consensus DNA sequence. Thymoquinone (5) was further identified as a nonpolar ingredient from the hexane extract of P. amboinicus to suppress the expression of lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-α). We then synthesized 2-alkylidenyl-4-cyclopentene-1,3-diones as the designed biomimetics of thymoquinone, and found that compounds 8a, 8b and 8d were more potent TNF-α inhibitors.

Oseltamivir hydroxamate and acyl sulfonamide derivatives as influenza neuraminidase inhibitors.

Tamiflu, the ethyl ester form of oseltamivir carboxylic acid (OC), is the first orally available anti-influenza drug for the front-line therapeutic option. In this study, the OC-hydroxamates, OC-sulfonamides and their guanidino congeners (GOC) were synthesized. Among them, an OC-hydroxamate 7d bearing an O-(2-indolyl)propyl substituent showed potent NA inhibition (IC50 = 6.4 nM) and good anti-influenza activity (EC50 = 60.1 nM) against the wild-type H1N1 virus. Two GOC-hydroxamates (9b and 9d) and one GOC-sulfonamide (12a) were active to the tamiflu-resistant H275Y virus (EC50 = 2.3-6.9 µM).

Dual-targeting compounds possessing enhanced anticancer activity via microtubule disruption and histone deacetylase inhibition.

Dual-targeting anticancer agents 4-29 are designed by combining the structural features of purine-type microtubule-disrupting compounds and HDAC inhibitors. A library of the conjugate compounds connected by appropriate linkers was synthesized and found to possess HDACs inhibitory activity and render microtubule fragmentation by activating katanin, a microtubule-severing protein. Among various zinc-binding groups, hydroxamic acid shows the highest inhibitory activity of Class I HDACs, which was also reconfirmed by three-dimensional quantitative structure-activity relationship (3D-QSAR) pharmacophore prediction. The purine-hydroxamate conjugates exhibit enhanced cytotoxicity against MDA-MB231 breast cancer cells, H1975 lung cancer cells, and various clinical isolated non-small-cell lung cancer cells with different epidermal growth factor receptor (EGFR) status. Pyridyl substituents could be used to replace the C2 and N9 phenyl moieties in the purine-type scaffold, which can help to improve the solubility under physiological conditions, thus increasing cytotoxicity. In mice treated with the purine-hydroxamate conjugates, the tumor growth rate was significantly reduced without causing toxic effects. Our study demonstrates the potential of the dual-targeting purine-hydroxamate compounds for cancer monotherapy.

Glutathione determines chronic myeloid leukemia vulnerability to an inhibitor of CMPK and TMPK.

Bcr-Abl transformation leads to chronic myeloid leukemia (CML). The acquirement of T315I mutation causes tyrosine kinase inhibitors (TKI) resistance. This study develops a compound, JMF4073, inhibiting thymidylate (TMP) and cytidylate (CMP) kinases, aiming for a new therapy against TKI-resistant CML. In vitro and in vivo treatment of JMF4073 eliminates WT-Bcr-Abl-32D CML cells. However, T315I-Bcr-Abl-32D cells are less vulnerable to JMF4073. Evidence is presented that ATF4-mediated upregulation of GSH causes T315I-Bcr-Abl-32D cells to be less sensitive to JMF4073. Reducing GSH biosynthesis generates replication stress in T315I-Bcr-Abl-32D cells that require dTTP/dCTP synthesis for survival, thus enabling JMF4073 susceptibility. It further shows that the levels of ATF4 and GSH in several human CML blast-crisis cell lines are inversely correlated with JMF4073 sensitivity, and the combinatory treatment of JMF4073 with GSH reducing agent leads to synthetic lethality in these CML blast-crisis lines. Altogether, the investigation indicates an alternative option in CML therapy.