The synthesis and biological evaluation of a library of autoinducer-antibiotic conjugates

Microbial resistance to antibiotics is a serious global health threat, and the discovery of new, safe and effective antibiotics is required urgently. A new class of antibiotics, namely sideophore-antibiotic conjugates, has shown promise in initial studies. Siderophores are used by bacteria for iron uptake, and so attaching antibiotics to them allows the antibiotic to be carried across cell membranes. This study investigated conjugates designed using a similar approach, but using bacterial autoinducers instead of siderophores. Autoinducers are required for coordination of bacterial behaviours and are involved in the control of swarming, virulence factor production and biofilm formation. The quorum sensing molecules produced by \textit{Pseudomonas aeruginosa} were chosen for investigation as \textit{P. aeruginosa} is a significant human pathogen which displays high resistance to many antibiotics and uses quorum sensing to coordinate its group behaviours. Ciprofloxacin and trimethoprim were chosen as the antibiotic partners. Ciprofloxacin is commonly used against \textit{P. aeruginosa} but resistance to it is developing, whereas \textit{P. aeruginosa} is inherently resistant to trimethoprim. It was hypothesised that the autoinducers would aid retention of the antibiotics in cells, hence increasing or restoring activity. An initial library was synthesised in two halves which were coupled together using a copper(I)-catalysed azide-alkyne cycloaddition. The autoinducers were functionalised with azide groups and the antibiotics (specifically ciprofloxacin and trimethoprim) were functionalised with alkynes. Two cleavable alkynyl ciprofloxacin derivatives were also included. A second set of compounds, namely homoserine lactone analogue-ciprofloxacin conjugates were then synthesised, building on the one known report of a conjugate of a quorum sensing modulator and an antibiotic. The most active conjugate found was a cleavable conjugate of homocysteine thiolactone (a homoserine lactone analogue) and ciprofloxacin. This compound showed enhanced antibacterial activity against \textit{P. aeruginosa} compared to ciprofloxacin, and \textit{P. aeruginosa} may develop less resistance towards it.EPSR

A 64 kDa sucrose binding protein is membrane-associated and tonoplast-localized in developing mung bean seeds

Sucrose binding proteins (SBPs) were predicted to be membrane-associated, but have been shown to localize in the lumen of protein storage vacuoles of various seeds. In this study, a new 64 kDa SBP has been identified from developing mung bean (Vigna radiata) seeds (here termed VrSBP1) via MS/MS analysis and N-terminal amino acid sequencing analysis and specific antibodies were generated using purified VrSBP1 proteins. Western blot analysis with the new VrSBP1 antibodies showed that, similar to most seed storage proteins, VrSBP1 proteins accumulated during seed development and were subsequently mobilized once the mung bean seeds germinated. Immunogold electron microscope (EM) studies on ultra-thin sections of high-pressure freezing/frozen substituted developing mung bean cotyledons demonstrated that VrSBP1 was localized specifically to the tonoplast of the protein storage vacuole and to the limiting membrane of a novel putative prevacuolar compartment. Biochemical and subcellular fractionation studies further demonstrated that VrSBP1 proteins were membrane-associated in developing mung beans, consistent with their tonoplast localization. This study thus shows convincing evidence of tonoplast-localization of a plant SBP for its future functional characterization and provides a model of studying non-integral membrane proteins associated with the tonoplasts in plant cells

Mechanistic insights into a BINOL-derived phosphoric acid-catalyzed asymmetric Pictet-Spengler reaction.

The reaction of tryptamine and (2-oxocyclohexyl)acetic acid can be catalyzed by 3,3'-bis(triphenylsilyl)-1,1'-bi-2-naphthol phosphoric acid to give an asymmetric β-carboline. This reaction was first studied by Holloway et al. ( Org. Lett. 2010 , 12 , 4720 - 4723 ), but their mechanistic work did not explain the high stereoselectivity achieved. This study uses density functional theory and hybrid quantum mechanics/molecular mechanics calculations to investigate this reaction and provide a model to explain its outcome. The step leading to diastereo- and enantioselectivity is an asymmetric Pictet-Spengler reaction involving an N-acyliminium ion bound to the catalyst in a bidentate fashion. This interaction occurs via hydrogen bonds between the two terminal oxygen atoms of the catalyst phosphate group and the hydrogen atoms at N and C2 of the substrate indole group. These bonds hold the transition structure rigidly and thus allow the catalyst triphenylsilyl groups to influence the enantioselectivity.We thank Girton College, Cambridge (research fellowship to M.N.G.), the EPSRC (studentship to M.N.G.), and Unilever for support.The is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/jo5028134

Molecular basis of histone tail recognition by human TIP5 PHD finger and bromodomain of the chromatin remodeling complex NoRC.

Tallant, C., et al., Structure 23, 80–92, January 6, 2015 http://dx.doi.org/10.1016/j.str.2014.10.017Binding of the chromatin remodeling complex NoRC to RNA complementary to the rDNA promoter mediates transcriptional repression. TIP5, the largest subunit of NoRC, is involved in recruitment to rDNA by interactions with promoter-bound TTF-I, pRNA, and acetylation of H4K16. TIP5 domains that recognize posttranslational modifications on histones are essential for recruitment of NoRC to chromatin, but how these reader modules recognize site-specific histone tails has remained elusive. Here, we report crystal structures of PHD zinc finger and bromodomains from human TIP5 and BAZ2B in free form and bound to H3 and/or H4 histones. PHD finger functions as an independent structural module in recognizing unmodified H3 histone tails, and the bromodomain prefers H3 and H4 acetylation marks followed by a key basic residue, KacXXR. Further low-resolution analyses of PHD-bromodomain modules provide molecular insights into their trans histone tail recognition, required for nucleosome recruitment and transcriptional repression of the NoRC complex.This work was supported by the UK Biotechnology and Biological Sciences Research Council (grants BB/G023123/1 David Phillips Fellowship to A.C. and BB/J001201/1 to A.C.) and a Federation of European Biochemical Societies short-term fellowship (04-11-12-10 to C.T.). We are grateful to Dr. Dimitri Y. Chirgadze of the Crystallographic X-Ray Facility at the Department of Biochemistry, University of Cambridge, and to the technical support at Diamond Light Source Synchrotron Facilities. We acknowledge support from the European Commission FP7 Programme under BioStruct-X (grant agreement 283570) for SAXS data collection at the EMBL (DESY). The SGC is a registered charity (No. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, the Canada Foundation for Innovation, the Canadian Institutes for Health Research, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda, and the Wellcome Trust (092809/Z/10/Z). E.V. is supported by a European Commission FP7 Marie Curie grant IDPbyNMR (contract 264257). P.F. is supported by a Welcome Trust Career Development Fellowship (095751/Z/11/Z)

A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5–Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response

Genome-Wide Identification, Functional Analysis and Expression Profiling of the Aux/IAA Gene Family in Tomato

Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components,among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk

Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes.Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a previously unknown transcriptional repressor family, and revealed their possible roles in plant growth and development

Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons

Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H+ symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level

The tomato SlIAA15 is involved in trichome formation and axillary shoot development

The Aux/IAA genes encode a large family of short-lived proteins known to regulate auxin signalling in plants. Functional characterization of SlIAA15, a member of the tomato (Solanum lycopersicum) Aux/IAA family, shows that the encoded protein acts as a strong repressor of auxin-dependent transcription. The physiological significance of SlIAA15 was addressed by a reverse genetics approach, revealing that SlIAA15 plays multiple roles in plant developmental processes. The SlIAA15 down-regulated lines display lower trichome number, reduced apical dominance with associated modified pattern of axillary shoot development, increased lateral root formation and decreased fruit set. Moreover, the leaves of SlIAA15-inhibited plants are dark green and thick, with larger pavement cells, longer palisade cells and larger intercellular space of spongy mesophyll cells. The SlIAA15-suppressed plants exhibit a strong reduction in type I, V and VI trichome formation, suggesting that auxin-dependent transcriptional regulation is required for trichome initiation. Concomitant with reduced trichome formation, the expression of some R2R3 MYB genes, putatively involved in the control of trichome differentiation, is altered. These phenotypes uncover novel and specialized roles for Aux/IAAs in plant developmental processes, clearly indicating that members of the Aux/IAA gene family in tomato perform both overlapping and specific functions