Activator-induced conformational changes regulate division-associated peptidoglycan amidases.

AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX-EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX-EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.

New Optically Active tert-Butylarylthiophosphinic Acids and Their Selenium Analogues as the Potential Synthons of Supramolecular Organometallic Complexes: Syntheses and Crystallographic Structure Determination.

The aim of the research described in this publication is two-fold. The first is a detailed description of the synthesis of a series of compounds containing a stereogenic heteroatom, namely the optically active P-stereogenic derivatives of tert-butylarylphoshinic acids bearing sulfur or selenium. The second is a detailed discussion dedicated to the determination of their structures by an X-ray analysis. Such a determination is needed when considering optically active hetero-oxophosphoric acids as new chiral solvating agents, precursors of new chiral ionic liquids, or ligands in complexes serving as novel organometallic catalysts.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Orychophragine D:A new 2-piperazinone fused 5-azacytosine type alkaloid with radioprotective activity from the seeds of Orychophragmus violaceus.

A new alkaloid, Orychophragine D (1), together with three known alkaloids, were isolated from the seeds of Orychophragmus violaceus. Orychophragine D represented the first example of 2-piperazinone fused 5-azacytosine skeleton. Their structures and absolute configurations were determined by spectroscopic analyses and X-ray crystallography. Compared to Ex-RAD, compound 1 exhibited a significant radioprotective activity on cell survival of irradiated HUVEC. In vivo experiments showed that 1 not only remarkably enhanced the survival of irradiated mice in 30 days, but also significantly promoted the recovery of the blood system of irradiated mice. These results suggested that 1 was valuable for further research as promising radioprotectors.

PK1 from Drosophila obscurin is an inactive pseudokinase with scaffolding properties.

Obscurins are large filamentous proteins with crucial roles in the assembly, stability and regulation of muscle. Characteristic of these proteins is a tandem of two C-terminal kinase domains, PK1 and PK2, that are separated by a long intrinsically disordered sequence. The significance of this conserved domain arrangement is unknown. Our study of PK1 from Drosophila obscurin shows that this is a pseudokinase with features typical of the CAM-kinase family, but which carries a minimalistic regulatory tail that no longer binds calmodulin or has mechanosensory properties typical of other sarcomeric kinases. PK1 binds ATP with high affinity, but in the absence of magnesium and lacks detectable phosphotransfer activity. It also has a highly diverged active site, strictly conserved across arthropods, that might have evolved to accommodate an unconventional binder. We find that PK1 interacts with PK2, suggesting a functional relation to the latter. These findings lead us to speculate that PK1/PK2 form a pseudokinase/kinase dual system, where PK1 might act as an allosteric regulator of PK2 and where mechanosensing properties, akin to those described for regulatory tails in titin-like kinases, might now reside on the unstructured interkinase segment. We propose that the PK1-interkinase-PK2 region constitutes an integrated functional unit in obscurin proteins.

Anti-inflammatory kaurane diterpenoids of Erythroxylum bezerrae.

Five unusual kaurane diterpenes, designated as bezerraditerpenes A-E (1-5), along with six known ones (6-11), were isolated from the hexane extract of the stems of Erythroxylum bezerrae. Their structures were elucidated based on the interpretation of the NMR spectroscopy, mass spectrometry, and X-ray diffraction analysis. The anti-inflammatory potential of the diterpenes 1-11 was screened through cellular viability and lipopolysaccharide (LPS)-induced nitric oxide (NO) production on murine macrophage-like cells RAW 264.7. Diterpene 6 (cauren-6ß-ol) showed potent cytotoxicity and increased ability to inhibit NO production. Diterpenes 1 (bezerraditerpene A), 2 (bezerraditerpene B), and 8 (ent-kaur-16-ene-3ß,15ß-diol) exhibited the same significant anti-inflammatory activity with NO CI50 inhibition (3.21-3.76 µM) without cytotoxicity, in addition to decreasing the levels of pro-inflammatory cytokines TNF-α and IL-6 in LPS-induced RAW264.7 cells.

Novel Sterically Crowded and Conformationally Constrained α-Aminophosphonates with a Near-Neutral pKa as Highly Accurate 31P NMR pH Probes. Application to Subtle pH Gradients Determination in Dictyostelium discoideum Cells.

In order to discover new 31P NMR markers for probing subtle pH changes (<0.2 pH unit) in biological environments, fifteen new conformationally constrained or sterically hindered α-aminophosphonates derived from diethyl(2-methylpyrrolidin-2-yl)phosphonate were synthesized and tested for their pH reporting and cytotoxic properties in vitro. All compounds showed near-neutral pKas (ranging 6.28−6.97), chemical shifts not overlapping those of phosphorus metabolites, and spectroscopic sensitivities (i.e., chemical shifts variation Δδab between the acidic and basic forms) ranging from 9.2−10.7 ppm, being fourfold larger than conventional endogenous markers such as inorganic phosphate. X-ray crystallographic studies combined with predictive empirical relationships and ab initio calculations addressed the inductive and stereochemical effects of substituents linked to the protonated amine function. Satisfactory correlations were established between pKas and both the 2D structure and pyramidalization at phosphorus, showing that steric crowding around the phosphorus is crucial for modulating Δδab. Finally, the hit 31P NMR pH probe 1b bearing an unsubstituted 1,3,2-dioxaphosphorinane ring, which is moderately lipophilic, nontoxic on A549 and NHLF cells, and showing pKa = 6.45 with Δδab = 10.64 ppm, allowed the first clear-cut evidence of trans-sarcolemmal pH gradients in normoxic Dictyostelium discoideum cells with an accuracy of <0.05 pH units.

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P4 and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.

Determination of Histone Methyltransferase Structure by Crystallography.

As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain.

Controlling Tautomerization in Pyridine-Fused Phosphorus-Nitrogen Heterocycles.

Inclusion of a second nitrogen atom in the aromatic core of phosphorus-nitrogen (PN) heterocycles results in unexpected tautomerization to a nonaromatic form. This tautomerization, initially observed in the solid state through X-ray crystallography, is also explained by computational analysis. We prepared an electron deficient analogue (2 e) with a fluorine on the pyridine ring and showed that the weakly basic pyridine resisted tautomerization, providing key insights to why the transformation occurs. To study the difference in solution vs. solid-state heterocycles, alkylated analogues that lock in the quinoidal tautomer were synthesized and their different 1 H NMR and UV/Vis spectra studied. Ultimately, we determined that all heterocycles are the aromatic tautomer in solution and all but 2 e switch to the quinoidal tautomer in the solid state. Better understanding of this transformation and under what circumstances it occurs suggest future use in a switchable on/off hydrogen-bond-directed receptor that can be tuned for complementary hydrogen bonding.

Integrative structural and functional analysis of human malic enzyme 3: A potential therapeutic target for pancreatic cancer.

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

Pentacyclic and hexacyclic cucurbitacins from Elaeocarpuspetiolatus.

Four undescribed cucurbitacins, designated as petiolaticins A-D, and four known cucurbitacins were isolated from the bark and leaves of Elaeocarpus petiolatus (Jack) Wall. Their chemical structures were elucidated based on detailed analyses of the NMR and MS data. The absolute configuration of petiolaticin A was also determined by X-ray diffraction analysis. Petiolaticin A represents a cucurbitacin derivative incorporating a 3,4-epoxyfuranyl-bearing side chain, while petiolaticin B possesses a furopyranyl unit fused to the tetracyclic cucurbitane core structure. Petiolaticins A, B, and D were evaluated in vitro against a panel of human breast, pancreatic, and colorectal cancer cell lines. Petiolaticin A exhibited the greatest cytotoxicity against the MDA-MB-468, MDA-MB-231, MCF-7, and SW48 cell lines (IC50 7.4, 9.2, 9.3, and 4.6 µM, respectively). Additionally, petiolaticin D, 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one, and 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one 3-O-ß-D-glucopyranoside were tested for their ability to inhibit cell entry of a pseudotyped virus bearing the hemagglutinin envelope protein of a highly pathogenic avian influenza virus. Petiolaticin D showed the highest inhibition (44.3%), followed by 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one (21.0%), and 16α,23α-epoxy-3ß,20ß-dihydroxy-10αH,23ßH-cucurbit-5,24-dien-11-one 3-O-ß-D-glucopyranoside showed limited inhibition (9.0%). These preliminary biological assays have demonstrated that petiolaticins A and D possess anticancer and antiviral properties, respectively, which warrant for further investigations.

Dimroth rearrangement-based synthesis of novel derivatives of [1,3]selenazolo[5,4-e][1,2,4]triazolo[1,5-c]pyrimidine as a new class of selenium-containing heterocyclic architecture.

As a part of our ongoing endeavor towards developing novel heterocyclic architectures, a number of novel Se-containing tricyclic heterocycles of the type [1,3]selenazolo[5,4-e][1,2,4]triazolo[1,5-c]pyrimidine have been synthesized through heteroannulation of a newly produced hydrazino derivative of selenazolo[4,5-d]pyrimidine with either orthoesters or carbon disulfide in pyridine followed by S-alkylation. Moreover, the multistep protocol employed in this investigation provides a new insight into the Dimroth rearrangement in both acidic and basic media as a means for the cyclocondensation of triazole on the selenazolopyrimidine framework leading to selenazolotriazolopyrimidines. The synthesis of new derivatives of novel selenazolotriazolopyrimidines via Dimroth rearrangement in both acidic and basic media is presented.

21,24-Cyclolanostanes revisited: Structural revision and biological evaluation.

Chemical fractionation of the EtOH extract of a medicinal macro fungus, Inonotus obliquus, afforded an array of lanostane-type triterpenoids (1-11) including two new ones (1 and 8). The structures of these compounds were determined on the basis of spectroscopic analyses, single crystal X-ray crystallography of 3-6 and biosynthetic considerations. With the confirmatory structural information provided by X-ray diffraction analysis in hand, several previously reported 21,24-cyclolanostanes, such as inonotsutriols A-C and (20R,21S,24S)-21,24-cyclopenta-3ß,21,25-trihydroxylanosta-8-ene, were structurally corrected. In addition, the NMR data of other types of 21,24-cyclo triterpenoids were also re-examined and structural revisions were thus suggested. Compounds 2, 6 and 8 showed significant cytostatic effects against a panel of tumor cell lines with IC50 values ranging from 7.80 to 18.5 µM. Further assays established that compound 2 exerted promising in vitro anti-breast cancer potential by inhibiting the proliferation and migration of 4T1 cells.

Inhibition of bacterial FMN transferase: A potential avenue for countering antimicrobial resistance.

Antibiotic resistance is a challenge for the control of bacterial infections. In an effort to explore unconventional avenues for antibacterial drug development, we focused on the FMN-transferase activity of the enzyme Ftp from the syphilis spirochete, Treponema pallidum (Ftp_Tp). This enzyme, which is only found in prokaryotes and trypanosomatids, post-translationally modifies proteins in the periplasm, covalently linking FMN (from FAD) to proteins that typically are important for establishing an essential electrochemical gradient across the cytoplasmic membrane. As such, Ftp inhibitors potentially represent a new class of antimicrobials. Previously, we showed that AMP is both a product of the Ftp_tp-catalyzed reaction and an inhibitor of the enzyme. As a preliminary step in exploiting this property to develop a novel Ftp_Tp inhibitor, we have used structural and solution studies to examine the inhibitory and enzyme-binding properties of several adenine-based nucleosides, with particular focus on the 2-position of the purine ring. Implications for future drug design are discussed.

Structure and ion-release mechanism of PIB-4-type ATPases.

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal PIB-4-ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

Heavy metals such as zinc and cobalt are toxic at high levels, yet most organisms need tiny amounts for their cells to work properly. As a result, proteins studded through the cell membrane act as gatekeepers to finetune import and export. These proteins are central to health and disease; their defect can lead to fatal illnesses in humans, and they also help bacteria infect other organisms. Despite their importance, little is known about some of these metal-export proteins. This is particularly the case for P_IB-4-ATPases, a subclass found in plants and bacteria and which includes, for example, a metal transporter required for bacteria to cause tuberculosis. Intricate knowledge of the three-dimensional structure of these proteins would help to understand how they select metals, shuttle the compounds in and out of cells, and are controlled by other cellular processes. To reveal this three-dimensional organisation, Grønberg et al. used X-ray diffraction, where high-energy radiation is passed through crystals of protein to reveal the positions of atoms. They focused on a type of PIB-4-ATPases found in bacteria as an example. The work showed that the protein does not contain the metal-binding regions seen in other classes of metal exporters; however, it sports unique features that are crucial for metal transport such as an adapted pathway for the transport of zinc and cobalt across the membrane. In addition, Grønberg et al. tested thousands of compounds to see if they could block the activity of the protein, identifying two that could kill bacteria. This better understanding of how PIB-4-ATPases work could help to engineer plants capable of removing heavy metals from contaminated soils, as well as uncover new compounds to be used as antibiotics.

Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1.

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase.

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

Perspectives on Fragment-based Drug Discovery: A Strategy Applicable to Diverse Targets.

Fragment-Based Drug Discovery (FBDD) is a strategy to develop potent lead molecules and is frequently used in drug discovery projects of the pharmaceutical industry. This method starts from identifying a small-molecule fragment, which usually binds weakly to the target and follows with a hit-to-lead step in which the fragment is grown into potent molecules that bind tightly to the target to affect its function. Quite a few drugs and compounds in clinical trials are developed using this approach, making FBDD a powerful strategy in drug discovery. FBDD can be applied to multiple targets and the hit rate in screening can be used in target druggability assessment. In this minireview, we provide a summary of the development of FBDD. In addition to giving a brief summary of the methods used in fragment screening, we highlight some methods that are critical in fragment growth. Biophysical methods and careful chemical modification of the fragments are the key elements in FBDD. We show several strategies that can be utilized in FBDD. We emphasize that NMR spectroscopy such as 19F-NMR and 1H-5N-HSQC experiment and X-ray crystallography are important in FBDD due to their roles in fragment screening and understanding the binding modes of the fragment hits, which provides a strategy for fragment growth.

Cell death-inducing activities via Hsp inhibition of the sesquiterpenes isolated from Valeriana fauriei.

Three new sesquiterpenes, valerianaterpenes I-III, and eight known compounds have been isolated from the methanol extract of the rhizomes and roots of Valeriana fauriei. The chemical structures of the three new sesquiterpenes were elucidated based on chemical and spectroscopic evidence. The absolute stereochemistry of valerianaterpene I was determined using X-ray crystallography. The cell death-inducing activity of isolated compounds alone or combination with Adriamycin (ADR) was observed by time-lapse cell imaging. Although the isolated compounds did not affect the number of mitotic entry cells and dead cells alone, kessyl glycol, kessyl glycol diacetate, and iso-teucladiol significantly increased the number of dead cells on ADR treated human cervical cancer cells. One of the mechanisms of cell death-inducing activity for the kessyl glycol acetate was suggested to be the inhibition of heat-shock protein 105 (Hsp105) expression level. This paper first deals with the naturally occurring compounds as Hsp105 inhibitor.