The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration.

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

Diversity in the Interaction of Amino Acid- and Peptide-Based Hydroxamic Acids with Some Platinum Group Metals in Solution.

Complexes that incorporate both ligand(s) and metal(s) exhibiting cytotoxic activity can especially be interesting to develop multifunctional drug molecules with desired activities. In this review, the limited number of solution results collected in our laboratory on the complexes of Pd(II) and two other platinum group metals-the half-sandwich type, [(η6-p-cym)Ru(H2O)3]2+, and [(η5-Cp*)Rh(H2O)3]2+-with hydroxamic acid derivatives of three amino acids, two imidazole analogues, and four small peptides are summarized and evaluated. Unlike the limited number of coordination sites of these metal ions (four and three for Pd(II) and the organometallic cations, respectively), the ligands discussed here offer a relatively high number of donor atoms as well as variation in their position within the ligands, resulting in a large versatility of the likely coordination modes. The review, besides presenting the solution equilibrium results, also discusses the main factors, such as (N,N) versus (O,O) chelate; size of chelate; amino-N versus imidazole-N; primary versus secondary hydroxamic function; differences between hydrolytic ability of the metal ions studied; and hydrolysis of the coordinated peptide hydroxamic acids in their Pd(II) complexes, which all determine the coordination modes present in the complexes formed in measurable concentrations in these systems. The options for the quantitative evaluation of metal binding effectivity and selectivity of the various ligands and the comparison with each other by using solution equilibrium data are also discussed.

Structure guided development of potent piperazine-derived hydroxamic acid inhibitors targeting falcilysin.

The protozoan parasite Plasmodium falciparum causes the most severe form of human malaria and is estimated to kill 400,000 people a year. The parasite infects and replicates in host red blood cells (RBCs), where it expresses an array of proteases to carry out multiple essential processes. We are investigating the function of falcilysin (FLN), a protease known to be required for parasite development in the RBC. We previously developed a piperazine-based hydroxamic acid scaffold to generate the first inhibitors of FLN, and the current study reports the optimization of the lead compound from that series. A range of substituents were tested at the N1 and N4 positions of the piperazine core, and inhibitors with significantly improved potency against purified FLN and cultured P. falciparum were identified. Computational studies were also performed to understand the mode of binding for these compounds, and predicted a binding model consistent with the biochemical data and the distinctive SAR observed at both the N1 and N4 positions.

Discovery of Novel Dihydropyrimidine and hydroxamic acid hybrids as potent Helicobacter pylori Urease inhibitors.

Two novel series of Dihydropyrimidine-hydroxamic acid hybrids (4a-4l and 5a-5l) were designed, synthesized and evaluated for in vitro Helicobacter pylori urease inhibition. In vitro enzyme inhibition screening led to the discovery of three potent urease inhibitors 2-[[4-(4-hydroxy phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy acetamide (4g), 2-[[4-(4-chloro phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy acetamide (4b) and 3-[[4-(3-methoxy phenyl)-6-oxo-1,6-dihydropyrimidine-2-yl]-amino]-N-hydroxy propanamide (5l). Compound 4g showed excellent urease inhibition with IC50 value of 14 ± 1 nM, indicated by its strong interactions with both metallic Ni++ ions, Gly279, His221, Ala365, Asp362, Asn168, Arg338 and His322 residues of the active site of urease. Further, compounds 4b and 5l displayed very good activity with IC50 value of 0.082 ± 0.004 µM and 0.14 ± 0.013 µM respectively compared to standard Acetohydroxamic acid (IC50 - 27.4 ± 1.2 µM). Kinetic studies revealed that a mixed inhibition with both competitive and non-competitive aspects is involved in the urease inhibition mechanism. The in vitro urease inhibition results were supported by molecular docking studies. Collectively, this study indicates that 4g could be considered as promising lead molecule that can be further developed as a potent drug molecule for the treatment of Helicobacter pylori caused gastritis for further studies.

Design, synthesis, and biological evaluation of hydroxamic acid-substituted 2,4-diaryl aminopyrimidines as potent EGFRT790M/L858R inhibitors for the treatment of NSCLC.

A series of 2,4-diarylaminopyrimidine derivatives bearing hydrophilic hydroxamic acids were designed and synthesized as potent EGFRT790M/L858R inhibitors. Among the derivatives synthesized, 10c (IC50 = 5.192 nM), 10j (IC50 = 10.35 nM), and 10o (IC50 = 0.3524 nM) exhibited higher potencies against EGFRT790/M/L858R compared to the known EGFR inhibitor AZD-9291 (IC50 = 20.80 nM). Moreover, 10j showed moderate activity against H1975 cells transfected with the EGFRT790M/L858R mutant, with an IC50 of 0.2113 µM over A431 (wild-type EGFR, SI = 47.3). In addition, 10j exhibited low toxicity in normal HBE cells (human bronchial epithelial cells, IC50 > 40 µΜ). Analysis of the mode of action indicated that 10j effectively induced apoptosis in H1975 cells by arresting the cells in the G2/M phase. Compound 10j also demonstrated efficacy in inhibiting tumor growth in a H1975 xenograft mouse model without losing body weight or killing the mice. Taken together, these results suggested that 10j might be a promising candidate for development as a potential treatment for NSCLC harboring the EGFRT790M/L858R mutation.

Investigation of the effect of different linker chemotypes on the inhibition of histone deacetylases (HDACs).

Histone Deacetylases (HDACs) are among the most attractive and interesting targets in anticancer drug discovery. The clinical relevance of HDAC inhibitors (HDACIs) is testified by four FDA-approved drugs for cancer treatment. However, one of the main drawbacks of these drugs resides in the lack of selectivity against the different HDAC isoforms, resulting in severe side effects. Thus, the identification of selective HDACIs represents an exciting challenge for medicinal chemists. HDACIs are composed of a cap group, a linker region, and a metal-binding group interacting with the catalytic zinc ion. While the cap group has been extensively investigated, less information is available about the effect of the linker on isoform selectivity. To this aim, in this work, we explored novel linker chemotypes to direct isoform selectivity. A small library of 25 hydroxamic acids with hitherto unexplored linker chemotypes was prepared. In vitro tests demonstrated that, depending on the linker type, some candidates selectively inhibit HDAC1 over HDAC6 isoform or vice versa. Docking calculations were performed to rationalize the effect of the novel linker chemotypes on biologic activity. Moreover, four compounds were able to increase the levels of acetylation of histone H3 or tubulin. These compounds were also assayed in breast cancer MCF7 cells to test their antiproliferative effect. Three compounds showed a significant reduction of cancer proliferation, representing valuable starting points for further optimization.

Population structure and pangenome analysis of Enterobacter bugandensis uncover the presence of blaCTX-M-55, blaNDM-5 and blaIMI-1, along with sophisticated iron acquisition strategies.

Enterobacter bugandensis is a recently described species that has been largely associated with nosocomial infections. We report the genome of a non-clinical E. bugandensis strain, which was integrated with publicly available genomes to study the pangenome and general population structure of E. bugandensis. Core- and whole-genome multilocus sequence typing allowed the detection of five E. bugandensis phylogroups (PG-A to E), which contain important antimicrobial resistance and virulence determinants. We uncovered several extended-spectrum ß-lactamases, including blaCTX-M-55 and blaNDM-5, present in an IncX replicon type plasmid, described here for the first time in E. bugandensis. Genetic context analysis of blaNDM-5 revealed the resemblance of this plasmid with other IncX plasmids from other bacteria from the same country. Three distinctive siderophore producing operons were found in E. bugandensis: enterobactin (ent), aerobactin (iuc/iut), and salmochelin (iro). Our findings provide novel insights on the lifestyle, physiology, antimicrobial, and virulence profiles of E. bugandensis.

Non-Hydroxamate Zinc-Binding Groups as Warheads for Histone Deacetylases.

Histone deacetylases (HDACs) remove acetyl groups from acetylated lysine residues and have a large variety of substrates and interaction partners. Therefore, it is not surprising that HDACs are involved in many diseases. Most inhibitors of zinc-dependent HDACs (HDACis) including approved drugs contain a hydroxamate as a zinc-binding group (ZBG), which is by far the biggest contributor to affinity, while chemical variation of the residual molecule is exploited to create more or less selectivity against HDAC isozymes or other metalloproteins. Hydroxamates have a propensity for nonspecificity and have recently come under considerable suspicion because of potential mutagenicity. Therefore, there are significant concerns when applying hydroxamate-containing compounds as therapeutics in chronic diseases beyond oncology due to unwanted toxic side effects. In the last years, several alternative ZBGs have been developed, which can replace the critical hydroxamate group in HDACis, while preserving high potency. Moreover, these compounds can be developed into highly selective inhibitors. This review aims at providing an overview of the progress in the field of non-hydroxamic HDACis in the time period from 2015 to present. Formally, ZBGs are clustered according to their binding mode and structural similarity to provide qualitative assessments and predictions based on available structural information.

Reduced Expression of Extracellular Matrix Proteins in the Heart and Kidneys of Rats with Endotoxemia under the Effect of Actinonin.

We studied modulation of the expression of extracellular matrix proteins under conditions of meprin inhibition in rats with LPS-induced endotoxemia. Endotoxemia increased the expression of type I, III, IV collagens and fibronectin in the renal tissue and type III and IV collagens in the heart. Meprin inhibitor actinonin reduced expression of both meprins and genes of extracellular matrix proteins, but the intensity of this effect in the heart and kidney was different. Inhibition of meprins in endotoxemia can prevent pathological remodeling of the extracellular matrix in the heart and kidney.

The Siderophore Synthetase IucA of the Aerobactin Biosynthetic Pathway Uses an Ordered Mechanism.

Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N6-acetyl-N6-hydroxylysine to the primary carboxylates of citrate. IucA and IucC are members of a family of non-ribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores, including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family were solved previously, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed a terreactant steady-state kinetic analysis and herein provide evidence for an ordered mechanism in which the chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with site-directed mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis.

OleD Loki as a Catalyst for Hydroxamate Glycosylation.

Herein we describe the ability of the permissive glycosyltransferase (GT) OleD Loki to convert a diverse set of >15 histone deacetylase (HDAC) inhibitors (HDACis) into their corresponding hydroxamate glycosyl esters. Representative glycosyl esters were subsequently evaluated in assays for cancer cell line cytotoxicity, chemical and enzymatic stability, and axolotl embryo tail regeneration. Computational substrate docking models were predictive of enzyme-catalyzed turnover and suggest certain HDACis may form unproductive, potentially inhibitory, complexes with GTs.

Discovery of a novel rhein-SAHA hybrid as a multi-targeted anti-glioblastoma drug.

Glioblastoma multiforme (GBM) is the most common malignant tumor of the central nervous system (CNS). Effective treatments remain limited. Therefore, novel chemotherapy drugs with high efficiency and few adverse effects are urgently needed. Histone deacetylase (HDAC) and serum and glucocorticoid-regulated protein kinase 1 (SGK1) are targets for the prevention and treatment of GBM. Rhein has antitumor and SGK1 suppression effects, although its biological activity is limited by poor bioavailability. To improve the drug-like properties of rhein, we constructed a novel rhein-hydroxyethyl hydroxamic acid derivative (SYSUP007), which combined rhein with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). In the present study, the human GBM cell lines, T98G, U87 and U251, were used to investigate the anticancer effects of SYSUP007 in vitro. We found that SYSUP007 was more effective in inhibiting glioma cell proliferation, invasion and migration in vitro compared with the effects of rhein and SAHA. We also confirmed that SYSUP007 increased the expression of Ac-K100 and NDRG1 (targets of HDAC and SGK1). The present study indicates the potential that SYSUP007, as a novel rhein and SAHA derivative, for development as an anti-cancer therapy.

Design, synthesis and biological evaluation of N-hydroxy-aminobenzyloxyarylamide analogues as novel selective κ opioid receptor antagonists.

Aminobenzyloxyarylamide derivatives 1a-i and 2a-t were designed and synthesized as novel selective κ opioid receptor (KOR) antagonists. The benzoyl amide moiety of LY2456302 was changed into N-hydroxybenzamide and benzisoxazole-3(2H)-one to investigate whether it could increase the binding affinity or selectivity for KOR. All target compounds were evaluated in radioligand binding assays for opioid receptor binding affinity. These efforts led to the identification of compound 1c (κ Ki = 179.9 nM), which exhibited high affinity for KOR. Moreover, the selectivity of KOR over MOR and DOR increased nearly 2-fold and 7-fold, respectively, compared with (±)LY2456302.

Screening for tyrosinase inhibitors from actinomycetes; identification of trichostatin derivatives from Streptomyces sp. CA-129531 and scale up production in bioreactor.

In the course of a primary screening of 614 microbial actinomycete extracts for the discovery of tyrosinase inhibitors, the EtOAc extract of the fermentation broth of the strain Streptomyces sp. CA-129531 isolated from a Martinique sample, exhibited in cell free and cell-based assays the most promising activity (IC50 value of 63 µg/mL). Scaled-up production in a bioreactor led to the isolation of one new trichostatic acid analogue, namely trichostatic acid B (1), along with six known trichostatin derivatives (2-7), four diketopiperazines (8-11), two butyrolactones (12-13) and one hydroxamic acid siderophore (14). Among them, trichostatin A (4) showed a Ki value of 6.1 µM and six times stronger anti-tyrosinase activity (IC50 2.18 µΜ) than kojic acid (IC50 14.07 µΜ) used as a positive control. Deoxytrichostatin A (6) displayed also strong inhibitory activity against tyrosinase (IC50 19.18 µΜ). Trichostatin A production in bioreactor started together with the exponential phase of growth (day 4) and the maximum concentration was reached at day 9 (2.67 ± 0.13 µg/mL). Despite the cytotoxicity of some individual components, the EtOAc extract showed no cytotoxic effect on HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, (IC50 >2.84 mg/mL) and against BG fibroblasts at the concentrations where the whitening effect was exerted, reassuring its safety and great tyrosinase inhibitory potential.

Heterologous expression of the trichostatin gene cluster and functional characterization of N-methyltransferase TsnB8.

Trichostatins are potent inhibitors of histone deacetylase (HDAC). In this work, a new trichostatin derivative isotrichostatin RK (1) and five known compounds trichostatin RK (2), JBIR-111 (3), 9179B (4), trichostatic acid (5) and trichostatin A (6) were isolated from marine-derived Streptomyces sp. SCSIO 40028. The biosynthetic gene cluster (tsnB) for trichostatins was identified from Streptomyces sp. SCSIO 40028 and validated by heterologous expression in Streptomyces lividans TK64. N-Methyltransferase TsnB8 was demonstrated to catalyze successive methyltransfer reactions by in vivo gene inactivation and in vitro enzyme assays.

Virtual Screening Approach for the Identification of Hydroxamic Acids as Novel Human Ecto-5'-Nucleotidase Inhibitors.

Ecto-5'-nucleotidase (ecto-5'-NT, CD73) is a zinc-binding metallophosphatase that plays a key role in extracellular purinergic pathways, being implicated in several physiological and pathophysiological processes, such as immune homeostasis, inflammation, and tumor progression. As such, it has been recognized as a promising biological target for many diseases, including cancer, infections, and autoimmune diseases. Despite its importance, so far only a few inhibitors of this target enzyme are known, most of which are not suitable as drug candidates. Here, we aimed to search for hydroxamic acid-containing compounds as potential human ecto-5'-NT inhibitors, since this group is known to be a strong zinc chelator. To this end, we performed a hierarchical virtual screening (VS) search consisting of three consecutive steps (filtering for compounds bearing a hydroxamic acid group, shape-based matching, and docking followed by visual inspection), which were applied to screen the ZINC-14 database ("all purchasable subset"). Out of 25 compounds selected by this VS protocol, 12 were acquired and further submitted to enzymatic assays for VS experimental validation. Four of them (i.e., 33.3%) were found to inhibit human ecto-5'-NT in the low micromolar range. The most potent one showed an IC50 value of 6.2 ± 1.0 µM. All identified inhibitors satisfy drug-like criteria and provide novel scaffolds to be explored in further hit-to-lead optimization steps. Furthermore, to the best of our knowledge, they are the first hydroxamic acid-containing inhibitors of human ecto-5'-NT described so far.

Uridine diphosphate glucuronosyltransferase 1A1.

The role that the phase-II reaction, glucuronidation, plays in the biotransformation of endo and xenobiotics is discussed with particular emphasis given to the UGT1A1 isoenzyme. This individual isoenzyme is responsible for both the mono and di-glucuronidation of bilirubin together with the glucuronidation of a number of xenobiotics of clinical interest (irinotecan, belinostat, atazanavir, pegvisomant).The review then discusses the roles that the various allelic variants of the UGT1A1 gene play in bilirubin metabolism and in particular how these allelic variants are involved in the clinical manifestation of the diseases of GS, CN1 and CN2.The review concludes with the roles that the UGT1A1*28 and UGT1A1*6 alleles play in adverse drug reactions (decreased glucuronidation of irinotecan, belinostat, atazanavir, pegvisomant) leading to increased exposure, reduced clearance and neutropenia (irinotecan, belinostat), increased risk for jaundice and hyperbilirubinaemia (atazanavir) and liver toxicity (pegvisomant) before discussing the future role of UGT1A1 in personalised medicine.

Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method.

D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered Escherichia coli strain. However, improvement and scaling-up of this process are hampered by the current methodology for D-DIBOA quantification, which is based on high-performance liquid chromatographic (HPLC), a time-consuming technique that requires expensive equipment and the use of environmentally unsafe solvents. In this work, we established and validated a rapid, simple, and sensitive spectrophotometric method for the quantification of the D-DIBOA produced by whole-cell biocatalysis, with limits of detection and quantification of 0.0165 and 0.0501 µmol·mL-1 respectively. This analysis takes place in only a few seconds and can be carried out using 100 µL of the sample in a microtiter plate reader. We performed several whole-cell biocatalysis strategies to optimize the process by monitoring D-DIBOA production every hour to keep control of both precursor and D-DIBOA concentrations in the bioreactor. These experiments allowed increasing the D-DIBOA production from the previously reported 5.01 mM up to 7.17 mM (43% increase). This methodology will facilitate processes such as the optimization of the biocatalyst, the scaling up, and the downstream purification.

Deciphering the Enzymatic Function of the Bovine Enteric Infection-Related Protein YfeJ from Salmonella enterica Serotype Typhimurium.

Non-typhoidal Salmonella can colonize the gastrointestinal system of cattle and can also cause significant food-borne disease in humans. The use of a library of single-gene deletions in Salmonella enterica serotype Typhimurium allowed identification of several proteins that are under selection in the intestine of cattle. STM2437 ( yfeJ) encodes one of these proteins, and it is currently annotated as a type I glutamine amidotransferase. STM2437 was purified to homogeneity, and its catalytic properties with a wide range of γ-glutamyl derivatives were determined. The catalytic efficiency toward the hydrolysis of l-glutamine was extremely weak with a kcat/ Km value of 20 M-1 s-1. γ-l-Glutamyl hydroxamate was identified as the best substrate for STM2437, with a kcat/ Km value of 9.6 × 104 M-1 s-1. A homology model of STM2437 was constructed on the basis of the known crystal structure of a protein of unknown function (Protein Data Bank entry 3L7N ), and γ-l-glutamyl hydroxamate was docked into the active site based on the binding of l-glutamine in the active site of carbamoyl phosphate synthetase. Acivicin was shown to inactivate the enzyme by reaction with the active site cysteine residue and the subsequent loss of HCl. Mutation of Cys91 to serine completely abolished catalytic activity. Inactivation of STM2437 did not affect the ability of this strain to colonize mice, but it inhibited the growth of S. enterica Typhimurium in bacteriologic media containing γ-l-glutamyl hydroxamate.

Structural and functional delineation of aerobactin biosynthesis in hypervirulent Klebsiella pneumoniae.

Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of Klebsiella pneumoniae (hvKP). In contrast to classical opportunistic strains of K. pneumoniae, hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent "super bug." Despite hvKP harboring four distinct siderophore operons, knocking out production of only aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. In vivo heterologous production and in vitro reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.