The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (– 153 ESEEKAWQKLCSL 165 –) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122–Trp159–Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57–Glu155–Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa , as well as the means to identify more potent ExoU inhibitors for future therapeutics.

Arginine methylation is a post-translational modification that is implicated in multiple biological functions including transcriptional regulation. The expression of protein arginine methyltransferases (PRMT) has been shown to be up-regulated in various cancers. PRMTs have emerged as attractive targets for the development of new cancer therapies. Here, we describe the identification of a natural compound, licochalcone A, as a novel, reversible and selective inhibitor of PRMT6. Since expression of PRMT6 is up-regulated in human breast cancers and is associated with oncogenesis, we used the human breast cancer cell line system to study the effect of licochalcone A treatment on PRMT6 activity, cell viability, cell cycle, and apoptosis. We demonstrated that licochalcone A is a non-S-adenosyl L-methionine (SAM) binding site competitive inhibitor of PRMT6. In MCF-7 cells, it inhibited PRMT6-dependent methylation of histone H3 at arginine 2 (H3R2), which resulted in a significant repression of estrogen receptor activity. Licochalcone A exhibited cytotoxicity towards human MCF-7 breast cancer cells, but not MCF-10A human breast epithelial cells, by up-regulating p53 expression and blocking cell cycle progression at G2/M, followed by apoptosis. Thus, licochalcone A has potential for further development as a therapeutic agent against breast cancer.

Aminopeptidase A (APA) is a membrane-bound monozinc aminopeptidase. In the brain, APA generates angiotensin III which exerts a tonic stimulatory effect on the control of blood pressure (BP) in hypertensive animals. The oral administration of RB150 renamed firibastat by WHO, an APA inhibitor prodrug, targeting only the S1 subsite, decreases BP in hypertensive patients from various ethnic origins. To identify new families of potent and selective APA inhibitors, we explored the organization of the APA active site, especially the S2′ subsite. By molecular modeling, docking, molecular dynamics simulations and site-directed mutagenesis, we revealed that Arg368 and Arg386, in the S2′ subsite of human APA established various types of interactions in major part with the P2′ residue but also with the P1′ residue of APA inhibitors, required for their nanomolar inhibitory potency. We also demonstrated an important role for Arg368 in APA catalysis, in maintaining the structural integrity of the GAMEN motif, a conserved sequence involved in exopeptidase specificity and optimal positioning of the substrate in monozinc aminopeptidases. This arginine together with the GAMEN motif are key players for the catalytic mechanism of these enzymes.

Despite being catalytically defective, pseudokinases are typically essential players of cellular signalling, acting as allosteric regulators of their active counterparts. Deregulation of a growing number of pseudokinases has been linked to human diseases, making pseudokinases therapeutic targets of interest. Pseudokinases can be dynamic, adopting specific conformations critical for their allosteric function. Interfering with their allosteric role, with small molecules that would lock pseudokinases in a conformation preventing their productive partner interactions, is an attractive therapeutic strategy to explore. As a well-known allosteric activator of epidermal growth factor receptor family members, and playing a major part in cancer progression, the pseudokinase HER3 is a relevant context in which to address the potential of pseudokinases as drug targets for the development of allosteric inhibitors. In this proof-of-concept study, we developed a multiplex, medium-throughput thermal shift assay screening strategy to assess over 100 000 compounds and identify selective small molecule inhibitors that would trap HER3 in a conformation which is unfavourable for the formation of an active HER2–HER3 heterodimer. As a proof-of-concept compound, AC3573 bound with some specificity to HER3 and abrogated HER2–HER3 complex formation and downstream signalling in cells. Our study highlights the opportunity to identify new molecular mechanisms of action interfering with the biological function of pseudokinases.

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signalling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilisation. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely ‘non-canonical’ substrates. Surprisingly, sequence interrogation of ∼300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signalling.

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2- O -sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2- O position of α- l -iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro , including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro . We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R 2 C 2 holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling.

Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to ‘normalise’ LRRK2 signalling in affected cell types. However, the development of such drugs requires validated assays for the analysis of target engagement and the assembly of a set of tools for interrogating LRRK2, and its substrates, both in vitro and in vivo . A new study published in the Biochemical Journal by Ito et al. establishes that a ‘Phos-tag’™-binding assay can be exploited to measure phosphorylation of a recently identified LRRK2 substrate (Ras-related protein in brain 10 (Rab10)), and to compare and contrast relative catalytic output from disease-associated LRRK2 mutants. Powerful in vivo chemical genetic approaches are also disclosed, in which the catalytic activity of LRRK2 is unequivocally linked to the extent of Rab10 phosphorylation and the effects of chemically distinct LRRK2 inhibitors are matched with on-target inhibition mechanisms mediated through LRRK2 and its substrate Rab10. These important findings should simplify the generic analysis of Rab10 phosphorylation in model biological systems and are likely to be applicable to other substrates of LRRK2 (or indeed other kinases) for which phospho-specific antibodies are either absent or unsatisfactory.

The mechanisms by which JAK2 is activated by the prevalent pseudokinase (JH2) V617F mutation in blood cancers remain elusive. Via structure-guided mutagenesis and transcriptional and functional assays, we identify a community of residues from the JH2 helix αC, SH2-JH2 linker and JH1 kinase domain that mediate V617F-induced activation. This circuit is broken by altering the charge of residues along the solvent-exposed face of the JH2 αC, which is predicted to interact with the SH2-JH2 linker and JH1. Mutations that remove negative charges or add positive charges, such as E596A/R, do not alter the JH2 V617F fold, as shown by the crystal structure of JH2 V617F E596A. Instead, they prevent kinase domain activation via modulation of the C-terminal residues of the SH2-JH2 linker. These results suggest strategies for selective V617F JAK2 inhibition, with preservation of wild-type function.

Historically, drugs used in the treatment of cancers also tend to cause damage to healthy cells while affecting cancer cells. Therefore, the identification of novel agents that act specifically against cancer cells remains a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis, which can cause cell death, by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cells. Therefore, targeting KIFC1 may provide novel insight into selective killing of cancer cells. In the present study, we identified a small-molecule KIFC1 inhibitor, SR31527, which inhibited microtubule (MT)-stimulated KIFC1 ATPase activity with an IC 50 value of 6.6 μM. By using bio layer interferometry technology, we further demonstrated that SR31527 bound directly to KIFC1 with high affinity ( K d =25.4 nM). Our results from computational modelling and saturation-transfer difference (STD)-NMR experiments suggest that SR31527 bound to a novel allosteric site of KIFC1 that appears suitable for developing selective inhibitors of KIFC1. Importantly, SR31527 prevented bipolar clustering of extra centrosomes in triple negative breast cancer (TNBC) cells and significantly reduced TNBC cell colony formation and viability, but was less toxic to normal fibroblasts. Therefore, SR31527 provides a valuable tool for studying the biological function of KIFC1 and serves as a potential lead for the development of novel therapeutic agents for breast cancer treatment.

Transforming growth factor-β (TGF-β) is a multifunctional cytokine implicated in many diseases, including tissue fibrosis and cancer. TGF-β mediates diverse biological responses by signalling through type I and II TGF-β receptors (TβRI and TβRII). We have previously identified CD109, a glycosylphosphatidylinositol (GPI)-anchored protein, as a novel TGF-β co-receptor that negatively regulates TGF-β signalling and responses and demonstrated that membrane-anchored CD109 promotes TGF-β receptor degradation via a SMAD7/Smurf2-mediated mechanism. To determine whether CD109 released from the cell surface (soluble CD109 or sCD109) also acts as a TGF-β antagonist, we determined the efficacy of recombinant sCD109 to interact with TGF-β and inhibit TGF-β signalling and responses. Our results demonstrate that sCD109 binds TGF-β with high affinity as determined by surface plasmon resonance (SPR) and cell-based radioligand binding and affinity labelling competition assays. SPR detected slow dissociation kinetics between sCD109 and TGF-β at low concentrations, indicating a stable and effective interaction. In addition, sCD109 antagonizes TGF-β-induced Smad2/3 phosphorylation, transcription and cell migration. Together, our results suggest that sCD109 can bind TGF-β, inhibit TGF-β binding to its receptors and decrease TGF-β signalling and TGF-β-induced cellular responses.

HIV-1 resistance to zidovudine [AZT (azidothymidine)] is associated with selection of the mutations M41L, D67N, K70R, L210W, T215F/Y and K219Q/E in RT (reverse transcriptase). These mutations decrease HIV-1 susceptibility to AZT by augmenting RT's ability to excise the chain-terminating AZT-MP (AZT-monophosphate) moiety from the chain-terminated DNA primer. Although AZT-MP excision occurs at the enzyme's polymerase active site, it is mechanistically distinct from the DNA polymerase reaction. Consequently, this activity represents a novel target for drug discovery, and inhibitors that target this activity may increase the efficacy of nucleoside/nucleotide analogues, and may help to delay the onset of drug resistance. In the present study, we have developed a FRET (Förster resonance energy transfer)-based high-throughput screening assay for the AZT-MP excision activity of RT. This assay is sensitive and robust, and demonstrates a signal-to-noise ratio of 3.3 and a Z’ factor of 0.69. We screened three chemical libraries (7265 compounds) using this assay, and identified APEX57219 {3,3′-[(3-carboxy-4-oxo-2,5-cyclohexadien-1-ylidene)methylene]bis[6-hydroxybenzoic acid]} as the most promising hit. APEX57219 displays a unique activity profile against wild-type and drug-resistant HIV-1 RT, and was found to inhibit virus replication at the level of reverse transcription. Mechanistic analyses revealed that APEX57219 blocked the interaction between RT and the nucleic acid substrate.

The tandem PHD (plant homeodomain) fingers of the CHD4 (chromodomain helicase DNA-binding protein 4) ATPase are epigenetic readers that bind either unmodified histone H3 tails or H3K9me3 (histone H3 trimethylated at Lys 9 ). This dual function is necessary for the transcriptional and chromatin remodelling activities of the NuRD (nucleosome remodelling and deacetylase) complex. In the present paper, we show that calixarene-based supramolecular hosts disrupt binding of the CHD4 PHD2 finger to H3K9me3, but do not affect the interaction of this protein with the H3K9me0 (unmodified histone H3) tail. A similar inhibitory effect, observed for the association of chromodomain of HP1γ (heterochromatin protein 1γ) with H3K9me3, points to a general mechanism of methyl-lysine caging by calixarenes and suggests a high potential for these compounds in biochemical applications. Immunofluorescence analysis reveals that the supramolecular agents induce changes in chromatin organization that are consistent with their binding to and disruption of H3K9me3 sites in living cells. The results of the present study suggest that the aromatic macrocyclic hosts can be used as a powerful new tool for characterizing methylation-driven epigenetic mechanisms.

Protein O -GlcNAcylation is an abundant, dynamic and reversible type of protein post-translational modification in animals that has been implicated in signalling processes linked to innate immunity, stress response, growth factor response, transcription, translation and proteosomal degradation. Only two enzymes, O -GlcNAc (O-linked N -acetylglucosamine) transferase and O -GlcNAcase, catalyse the reversible addition of the O -GlcNAc residue to over 1000 target proteins in the human cell. Recent advances in our understanding of the structures and mechanisms of these enzymes have resulted in the development of potent and selective inhibitors. The present review gives an overview of these inhibitors and how they have been used on cell lines, primary cells and animals to modulate O -GlcNAc levels and study the effects on signal transduction.

The multifunctional nature of Alzheimer's disease calls for MTDLs (multitarget-directed ligands) to act on different components of the pathology, like the cholinergic dysfunction and amyloid aggregation. Such MTDLs are usually on the basis of cholinesterase inhibitors (e.g. tacrine or huprine) coupled with another active molecule aimed at a different target. To aid in the design of these MTDLs, we report the crystal structures of hAChE (human acetylcholinesterase) in complex with FAS-2 (fasciculin 2) and a hydroxylated derivative of huprine (huprine W), and of hBChE (human butyrylcholinesterase) in complex with tacrine. Huprine W in hAChE and tacrine in hBChE reside in strikingly similar positions highlighting the conservation of key interactions, namely, π-π/cation-π interactions with Trp 86 (Trp 82 ), and hydrogen bonding with the main chain carbonyl of the catalytic histidine residue. Huprine W forms additional interactions with hAChE, which explains its superior affinity: the isoquinoline moiety is associated with a group of aromatic residues (Tyr 337 , Phe 338 and Phe 295 not present in hBChE) in addition to Trp 86 ; the hydroxyl group is hydrogen bonded to both the catalytic serine residue and residues in the oxyanion hole; and the chlorine substituent is nested in a hydrophobic pocket interacting strongly with Trp 439 . There is no pocket in hBChE that is able to accommodate the chlorine substituent.

Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of kinase inhibition across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using biochemical assays for inhibitory activity against 234 active human kinases and kinase complexes, representing all branches of the kinome tree. For our study we employed 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important as therapeutic targets and/or signal transduction regulators. Hierarchical clustering of these benchmark kinase inhibitors on the basis of their kinome activity profiles illustrates how they relate to chemical structure similarities and provides new insights into inhibitor specificity and potential applications for probing new targets. Using this broad dataset, we provide a framework for assessing polypharmacology. We not only discover likely off-target inhibitor activities and recommend specific inhibitors for existing targets, but also identify potential new uses for known small molecules.

miRNAs (microRNAs) are short non-coding RNAs that can regulate gene expression in cancer development, which makes them valuable targets for therapeutic intervention. In the present study we report on an approach that can not only arrest the functions of mature miRNAs by binding to them, but it can also induce the ‘mis-processing’ of the target miRNA, producing a non-functional truncated miRNA. This approach involves generating an expression construct that produces an RNA fragment with 16 repeat sequences. The construct is named miR-Pirate ( m iRNA- i nteracting R NA- p roducing i mperfect R NA a nd t angling e ndogenous miRNA). The transcript of the construct contained mismatches to the seed region, and thus it would not target the potential targets of the miRNA under study. The homology of the construct is sufficiently high, allowing the transcript to block miRNA functions. The functions of the construct were validated in cell cultures, in tumour formation assays and in transgenic mice stably expressing this construct. To explore the possibility of adopting this approach in gene therapy, we transfected cells with synthetic miR-Pirate and obtained the results we expected. The miR-Pirate , expressed by the construct or synthesized chemically, was found to be able to specifically pirate and silence a mature miRNA through its dual roles and thus could be clinically applied for miRNA intervention.

Jaundice or hyperbilirubinaemia is a common complication of sepsis. UGT1A1 (UDP-glucuronosyltransferase 1A1) is a critical gene for bilirubin metabolism and irinotecan detoxification. However, the molecular pathogenesis of hyperbilirubinaemia during inflammation needs to be further clarified. Human hepatic UGT1A1 expression was analysed by RT (reverse transcription)–PCR, qRT-PCR (quantitative real-time PCR) and Western blotting in response to LPS (lipopolysaccharide) stimulation. Transcription regulatory elements in the upstream promoter region of the human UGT1A1 gene were determined using EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation). The important role of the transcription regulatory element was examined using a luciferase assay, and was determined by qRT-PCR using a transcription factor activation inhibitor. LPS down-regulated the UGT1A1 mRNA expression in human hepatoma cell lines. A newly identified NF-κB (nuclear factor κB)-binding site was located on the upstream promoter region (−725/−716) of the human UGT1A1 gene. LPS-induced NF-κB activation and specific binding to the NF-κB-binding site can suppress human UGT1A1 promoter activity and human UGT1A1 expression. We demonstrated that LPS mediates the suppression of human UGT1A1 expression through specific binding of NF-κB to this newly identified NF-κB-binding site in the upstream promoter of the human UGT1A1 gene. The present study may partly explain the molecular pathogenesis of inflammation-associated hyperbilirubinaemia.

Erlotinib and gefitinib, tyrosine kinase inhibitors used to block EGFR (epidermal growth factor receptor) signalling in cancer, are thought to bind only the active conformation of the EGFR-TKD (tyrosine kinase domain). Through parallel computational and crystallographic studies, we show in the present study that erlotinib also binds the inactive EGFR-TKD conformation, which may have significant implications for its use in EGFR-mutated cancers.

GlmU is a bifunctional enzyme with acetyltransferase and uridyltransferase activities, and is essential for the biosynthesis of the bacterial cell wall. Inhibition results in a loss of cell viability. GlmU is therefore considered a potential target for novel antibacterial agents. A HTS (high-throughput screen) identified a series of aminoquinazolines with submicromolar potency against the uridyltransferase reaction. Biochemical and biophysical characterization showed competition with UTP binding. We determined the crystal structure of a representative aminoquinazoline bound to the Haemophilus influenzae isoenzyme at a resolution of 2.0 Å. The inhibitor occupies part of the UTP site, skirts the outer perimeter of the GlcNAc1-P ( N -acetylglucosamine-1-phosphate) pocket and anchors a hydrophobic moiety into a lipophilic pocket. Our SAR (structure–activity relationship) analysis shows that all of these interactions are essential for inhibitory activity in this series. The crystal structure suggests that the compound would block binding of UTP and lock GlmU in an apo-enzyme-like conformation, thus interfering with its enzymatic activity. Our lead generation effort provides ample scope for further optimization of these compounds for antibacterial drug discovery.

Chitinolytic β- N -acetyl- D -hexosaminidase is a branch of the GH20 (glycoside hydrolase family 20) β- N -acetyl- D -hexosaminidases that is only distributed in insects and micro-organisms, and is therefore a potential target for the action of insecticides. PUGNAc [ O -(2-acetamido-2-deoxy- D -glucopyransylidene)-amino- N -phenylcarbamate] was initially identified as an inhibitor against GH20 β- N -acetyl- D -hexosaminidases. So far no crystal structure of PUGNAc in complex with any GH20 β- N -acetyl- D -hexosaminidase has been reported. We show in the present study that the sensitivities of chitinolytic β- N -acetyl- D -hexosaminidases towards PUGNAc can vary by 100-fold, with the order being OfHex1 ( Ostrinia furnacalis β- N -acetyl- D -hexosaminidase)<SmCHB ( Serratia marcescens chitobiase)<SpHex ( Streptomyces plicatus β- N -acetyl- D -hexosaminidase). To explain this difference, the crystal structures of wild-type OfHex1 as well as mutant OfHex1(V327G) in complex with PUGNAc were determined at 2.0 Å (1 Å=0.1 nm) and 2.3 Å resolutions and aligned with the complex structures of SpHex and SmCHB. The results showed that the sensitivities of these enzymes to PUGNAc were determined by the active pocket size, with OfHex1 having the largest but narrowest entrance, whereas SpHex has the smallest entrance, suitable for holding the inhibitor, and SmCHB has the widest entrance. By widening the size of the active pocket entrance of OfHex1 through replacing the active site Val 327 with a glycine residue, the sensitivity of OfHex1 to PUGNAc became similar to that of SmCHB. The structural differences among chitinolytic β- N -acetyl- D -hexosaminidases leading to different sensitivities to PUGNAc may be useful for developing species-specific pesticides and bactericides.

ATP binding to the ϵ subunit of F 1 -ATPase, a soluble subcomplex of TF o F 1 (F o F 1 -ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F 1 -ATPase activity by stabilizing the compact, ATPase-active, form of the ϵ subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282 , 37618–37623]. In the present study, we report how ATP binding to the ϵ subunit affects ATPase and H + pumping activities in the holoenzyme TF o F 1 . Wild-type TF o F 1 showed significant H + pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ϵ subunit, did not support efficient H + pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H + pumping activities. Similar uncoupling was observed when TF o F 1 contained an ATP-binding-deficient ϵ subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ϵ subunit induced by ATP binding was required to couple ATPase and H + pumping activities in TF o F 1 unless the ϵ subunit was in its extended-state conformation. The present study reveals a novel role of the ϵ subunit as an ATP-sensitive regulator of the coupling of ATPase and H + pumping activities of TF o F 1 .

The interaction of human topoisomerase I and erybraedin C, a pterocarpan purified from the plant Bituminaria bituminosa , that was shown to have an antitumour activity, was investigated through enzymatic activity assays and molecular docking procedures. Erybraedin C is able to inhibit both the cleavage and the religation steps of the enzyme reaction. In both cases, pre-incubation of the drug with the enzyme is required to produce a complete inhibition. Molecular docking simulations indicate that, when interacting with the enzyme alone, the preferential drug-binding site is localized in proximity to the active Tyr 723 residue, with one of the two prenilic groups close to the active-site residues Arg 488 and His 632 , essential for the catalytic reaction. When interacting with the cleavable complex, erybraedin C interacts with both the enzyme and DNA in a way similar to that found for topotecan. This is the first example of a natural compound able to act on both the cleavage and religation reaction of human topoisomerase I.

The Plasmodium falciparum cysteine peptidases FP-2 (falcipain-2) and FP-3 (falcipain-3), members of the papain-like CAC1 family, are essential haemoglobinases and are therefore potential anti-malarial drug targets. To facilitate a rational drug discovery programme, in the current study we analysed the synthetic substrate and model inhibitor profiles of FP-2 and FP-3 as well as BP-2 (berghepain-2), an orthologue from the rodent parasite Plasmodium berghei . With respect to substrate catalysis, FP-2 exhibited a promiscuous substrate profile based around a consensus non-primeside motif, FP-3 was somewhat more restricted and BP-2 was comparatively specific. Substrate turnover for FP-2 was driven by a basic or acidic P1 residue, whereas for FP-3 turnover occurred predominately through a basic P1 residue only, and for BP-2, turnover was again mainly through a basic P1 residue for some motifs and surprisingly a glycine in the P1 position for other motifs. Within these P1 binding elements, additional recognition motifs were observed with subtle nuances that switched substrate turnover on or off through specific synergistic combinations. The peptidases were also profiled against reversible and irreversible cysteine peptidase inhibitors. The results re-iterated the contrasting kinetic behaviour of each peptidase as observed through the substrate screens. The results showed that the substrate and inhibitor preferences of BP-2 were markedly different from those of FP-2 and FP-3. When FP-2 and FP-3 were compared to each other they also displayed similarities and some significant differences. In conclusion, the in vitro data highlights the current difficulties faced by a peptidase directed anti-malarial medicinal chemistry programme where compounds need to be identified with potent activity against at least three peptidases, each of which displays distinct biochemical traits.

Many viral proteins undergo proteolytic processing events that are required for virus infection and virion assembly. In this issue of Biochemical Journal , Strongin and co-workers report that the NS3 protease from West Nile virus unexpectedly cleaves certain substrates at pairs of basic residues, a specificity that resembles that of the furin-like PCs (proprotein convertases). This led to the demonstration that furin/PC inhibitors containing poly( D -arginine) are also potent inhibitors of NS3, and that anthrax toxin protective antigen and myelin basic protein are potential NS3 substrates. Structural modelling based on Dengue virus NS3 provided a possible rationale for the observed cleavage specificity of West Nile virus NS3.

HIV-1 protease is an effective target for the design of drugs against AIDS. To help this process of drug design, three-dimensional structures have been determined of complexes between HIV-1 protease and a variety of transition-state analogue inhibitors. The true transition state, however, has not been structurally characterized. The crystal structure of the C95M/C1095A HIV-1 protease tethered dimer shows a distinctive feature in which the two flaps of the enzyme are in a ‘closed conformation’ even in the unliganded state. This unique feature has been utilized here to study the structure of HIV-1 protease complexed to an oligopeptide substrate of amino acid sequence His-Lys-Ala-Arg-Val-Leu*NPhe-Glu-Ala-Nle-Ser (where* denotes the cleavage site, and NPhe and Nle denote p -nitrophenylalanine and norleucine residues respectively). The X-ray structure of the complex refined against 2.03 Å (0.203 nm) resolution synchrotron data shows that the substrate is trapped as a tetrahedral reaction intermediate in the crystal. The hydrogen-bonding interactions between the reaction intermediate and the catalytic aspartates are different from those observed previously using transition-state analogues. The reaction intermediate did not dissociate to release the products, possibly due to the inflexibility introduced in the flaps when the enzyme is packed inside crystals.

Bulgecin A, a sulphonated N -acetyl- D -glucosamine unit linked to a 4-hydroxy-5-hydroxymethylproline ring by a β-glycosidic linkage, is a novel type of inhibitor for binuclear metallo-β-lactamases. Using steady-state kinetic analysis with nitrocefin as the β-lactam substrate, bulgecin A competitively inhibited the metallo-β-lactamase BceII from Bacillus cereus in its two-zinc form, but failed to inhibit when the enzyme was in the single-zinc form. The competitive inhibition was restored by restoring the second zinc ion. The single-zinc metallo-β-lactamase from Aeromonas veronii bv. sobria , ImiS, was not inhibited by bulgecin A. The tetrameric L1 metallo-β-lactamase from Stenotrophomonas maltophilia was subject to partial non-competitive inhibition, which is consistent with a kinetic model in which the enzyme bound to inhibitor retains catalytic activity. Docking experiments support the conclusion that bulgecin A co-ordinates to the zinc II site in metallo-β-lactamases via the terminal sulphonate group on the sugar moiety.

We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC 50 4.6 μM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC 50 values of 2.7 and 21 μM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 μM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr 308 and Ser 473 , reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.

Staphopain A is a staphylococcal cysteine protease. Genes encoding staphopain A and its specific inhibitor, staphostatin A, are localized in an operon. Staphopain A is an important staphylococcal virulence factor. It is difficult to perform studies on its interaction with other proteins due to problems in obtaining a sufficient amount of the enzyme from natural sources. Therefore efforts were made to produce a recombinant staphopain A. Sequences encoding the mature form of staphopain A and staphostatin A were PCR-amplified from Staphylococcus aureus genomic DNA and cloned into different compatible expression vectors. Production of staphopain A was observed only when the enzyme was co-expressed together with its specific inhibitor, staphostatin A. Loss of the function mutations introduced within the active site of staphopain A causes the expression of the inactive enzyme. Mutations within the reactive centre of staphostatin A result in abrogation of production of both the co-expressed proteins. These results support the thesis that the toxicity of recombinant staphopain A to the host is due to its proteolytic activity. The coexpressed proteins are located in the insoluble fraction. Ni 2+ -nitrilotriacetate immobilized metal-affinity chromatography allows for an efficient and easy purification of staphopain A. Our optimized refolding parameters allow restoration of the native conformation of the enzyme, with yields over 10-fold higher when compared with isolation from natural sources.

The death morphology commonly known as apoptosis results from a post-translational pathway driven largely by specific limited proteolysis. In the last decade the structural basis for apoptosis regulation has moved from nothing to ‘quite good’, and we now know the fundamental structures of examples from the initiator phase, the pre-mitochondrial regulator phase, the executioner phase, inhibitors and their antagonists, and even the structures of some substrates. The field is as well advanced as the best known of proteolytic pathways, the coagulation cascade. Fundamentally new mechanisms in protease regulation have been disclosed. Structural evidence suggests that caspases have an unusual catalytic mechanism, and that they are activated by apparently unrelated events, depending on which position in the apoptotic pathway they occupy. Some naturally occurring caspase inhibitors have adopted classic inhibition strategies, but other have revealed completely novel mechanisms. All of the structural and mechanistic information can, and is, being applied to drive therapeutic strategies to combat overactivation of apoptosis in degenerative disease, and underactivation in neoplasia. We present a comprehensive review of the caspases, their regulators and inhibitors from a structural and mechanistic point of view, and with an aim to consolidate the many threads that define the rapid growth of this field.

Triclosan, a known antibacterial, acts by inhibiting enoyl-ACP (acyl-carrier protein) reductase (ENR), a key enzyme of the type II fatty acid synthesis (FAS) system. Plasmodium falciparum , the human malaria-causing parasite, harbours the type II FAS; in contrast, its human host utilizes type I FAS. Due to this striking difference, ENR has emerged as an important target for the development of new antimalarials. Modelling studies, and the crystal structure of P. falciparum ENR, have highlighted the features of ternary complex formation between the enzyme, triclosan and NAD + [Suguna, A. Surolia and N. Surolia (2001) Biochem. Biophys. Res. Commun. 283 , 224–228; Perozzo, Kuo, Sidhu, Valiyaveettil, Bittman, Jacobs, Fidock, and Sacchettini (2002) J. Biol. Chem. 277 , 13106–13114; and Swarnamukhi, Kapoor, N. Surolia, A. Surolia and Suguna (2003) PDB1UH5]. To address the issue of the importance of the residues involved in strong specific and stoichiometric binding of triclosan to P. falciparum ENR, we mutated the following residues: Ala-217, Asn-218, Met-281, and Phe-368. The affinity of all the mutants was reduced for triclosan as compared with the wild-type enzyme to different extents. The most significant mutation was A217V, which led to a greater than 7000-fold decrease in the binding affinity for triclosan as compared with wild-type PfENR. A217G showed only 10-fold reduction in the binding affinity. Thus, these studies point out significant differences in the triclosan-binding region of the P. falciparum enzyme from those of its bacterial counterparts.

The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikreins were assayed with the bradykinin-containing synthetic peptides Abz-MTEMARRPPGFSPFRSVTVQNH 2 (where Abz stands for o -aminobenzoyl) and Abz-MTSVIRRPPGFSPFRAPRV-NH 2 , which correspond to fragments Met 374 -Gln 393 and Met 375 -Val 393 of mouse and rat LMWKs (low-molecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1- and rK1-mediated hydrolysis of Arg–Arg and Arg–Ser (or Arg–Ala) peptide bonds. However, owing to preferential hydrolysis of Phe–Arg compared with the Arg–Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg 9 ]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH 2 ). The formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N- or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe–Arg bond, confirming the potential des-[Arg 9 ]bradykinin-releasing activity of hK1 on rat kininogen. The proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg–Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S 1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg 9 ]bradykinin. For further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.

The activities of PP1 (protein phosphatase 1), a principal cellular phosphatase that reverses serine/threonine protein phosphorylation, can be altered by inhibitors whose activities are themselves regulated by phosphorylation. We now describe a novel PKC (protein kinase C)-dependent PP1 inhibitor, namely GBPI (gut and brain phosphatase inhibitor). The shorter mRNA that encodes this protein, GBPI-1, is expressed in brain, stomach, small intestine, colon and kidney, whereas a longer GBPI-2 splice variant mRNA is found in testis. Human GBPI-1 mRNA encodes a 145-amino-acid, 16.5 kDa protein with pI 7.92. GBPI contains a consensus PP1-binding motif at residues 21–25 and consensus sites for phosphorylation by enzymes, including PKC, PKA (protein kinase A or cAMP-dependent protein kinase) and casein kinase II. Recombinant GBPI-1-fusion protein inhibits PP1 activity with IC 50 =3 nM after phosphorylation by PKC. Phospho-GBPI can even enhance PP2A activity by >50% at submicromolar concentrations. Non-phosphorylated GBPI-1 is inactive in both assays. Each of the mutations in amino acids located in potential PP1-binding sequences, K21E+K22E and W25A, decrease the ability of GBPI-1 to inhibit PP1. Mutations in the potential PKC phosphoacceptor site T58E also dramatically decrease the ability of GBPI-1 to inhibit PP1. Interestingly, when PKC-phosphorylated GBPI-1 is further phosphorylated by PKA, it no longer inhibits PP1. Thus, GBPI-1 is well positioned to integrate PKC and PKA modulation of PP1 to regulate differentially protein phosphorylation patterns in brain and gut. GBPI, its closest family member CPI (PKC-potentiated PP1 inhibitor) and two other family members, kinase-enhanced phosphatase inhibitor and phosphatase holoenzyme inhibitor, probably modulate integrated control of protein phosphorylation states in these and other tissues.

Telomere maintenance is essential for the continued proliferation of dividing cells, and is implicated in chromosome stability and cell immortalization. Telomerase activity allows cells to maintain their telomeric DNA and contributes to the indefinite replicative capacity of cancer cells. Telomerase is expressed in most cancer cells, but not in normal somatic cells, suggesting that telomerase is an attractive target for cancer chemotherapy. Here we screened a chemical library for inhibition of human telomerase, and identified 2,3,7-trichloro-5-nitroquinoxaline (TNQX) as a potent inhibitor. TNQX showed a potent inhibitory effect, with 50% inhibition at ~1.4 μM, and did not inhibit DNA and RNA polymerases, including retroviral reverse trancriptase. A series of enzyme kinetic experiments suggested that TNQX is a mixed-type non-competitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate (TS) primer and the dNTPs. Long-term cultivation of the MCF7 cell line with a drug concentration that did not cause acute cytotoxicity resulted in progressive telomere erosion followed by an increased incidence of chromosome abnormalities and induction of the senescence phenotype. The results presented here indicate that TNQX is a highly potent and selective anti-telomerase agent with good potential for further development as a promising anti-cancer agent.

Transcriptional regulation in eukaryotes occurs within a chromatin setting, and is strongly influenced by the post-translational modification of histones, the building blocks of chromatin, such as methylation, phosphorylation and acetylation. Acetylation is probably the best understood of these modifications: hyperacetylation leads to an increase in the expression of particular genes, and hypoacetylation has the opposite effect. Many studies have identified several large, multisubunit enzyme complexes that are responsible for the targeted deacetylation of histones. The aim of this review is to give a comprehensive overview of the structure, function and tissue distribution of members of the classical histone deacetylase (HDAC) family, in order to gain insight into the regulation of gene expression through HDAC activity. SAGE (serial analysis of gene expression) data show that HDACs are generally expressed in almost all tissues investigated. Surprisingly, no major differences were observed between the expression pattern in normal and malignant tissues. However, significant variation in HDAC expression was observed within tissue types. HDAC inhibitors have been shown to induce specific changes in gene expression and to influence a variety of other processes, including growth arrest, differentiation, cytotoxicity and induction of apoptosis. This challenging field has generated many fascinating results which will ultimately lead to a better understanding of the mechanism of gene transcription as a whole.

The catalytic subunit of type 1 Ser/Thr protein phosphatases (PP1c) forms complexes with many proteins that target it to particular subcellular locations and regulate its activity towards specific substrates. We report the identification of a Drosophila orthologue of nuclear inhibitor of PP1 (NIPP1Dm) through interaction with PP1c in the yeast two-hybrid system. NIPP1Dm shares many properties with mammalian NIPP1 including inhibition of PP1c in vitro , binding to RNA and PP1c, and localization to nuclear speckles. However, the mechanism controlling interaction of PP1c with NIPP1 is not conserved in Drosophila. NIPP1 can function independently of PP1c as a splicing factor, but the relative importance of this function is unknown. Over-expression of NIPP1Dm in Drosophila is cell-lethal in a range of tissues and developmental stages. The effects of ectopic NIPP1Dm are suppressed by co-expression of PP1c, indicating that the only effect of ectopic NIPP1Dm is to affect PP1c function. Co-expression of NIPP1Dm and PP1c does not have any detectable physiological effect in vivo , suggesting that the NIPP1Dm—PP1c holoenzyme is not normally limiting in Drosophila . These data show that NIPP1Dm and PP1c interact in vivo and suggest that NIPP1's role as a phosphatase regulator is conserved in Drosophila .

Luteolin, a naturally occurring flavonoid, is abundant in our daily dietary intake. It exhibits a wide spectrum of pharmacological properties, but little is known about its biochemical targets other than the fact that it induces topoisomerase II-mediated apoptosis. In the present study, we show that luteolin completely inhibits the catalytic activity of eukaryotic DNA topoisomerase I at a concentration of 40 μ M, with an IC 50 of 5 μ M. Preincubation of enzyme with luteolin before adding a DNA substrate increases the inhibition of the catalytic activity (IC 50 = 0.66 μ M). Treatment of DNA with luteolin before addition of topoisomerase I reduces this inhibitory effect. Subsequent fluorescence tests show that luteolin not only interacts directly with the enzyme but also with the substrate DNA, and intercalates at a very high concentration (>250 μ M) without binding to the minor groove. Direct interaction between luteolin and DNA does not affect the assembly of the enzyme–DNA complex, as evident from the electrophoretic mobility-shift assays. Here we show that the inhibition of topoisomerase I by luteolin is due to the stabilization of topoisomerase-I DNA-cleavable complexes. Hence, luteolin is similar to camptothecin, a class I inhibitor, with respect to its ability to form the topoisomerase I-mediated ‘cleavable complex'. But, unlike camptothecin, luteolin interacts with both free enzyme and substrate DNA. The inhibitory effect of luteolin is translated into concanavalin A-stimulated mouse splenocytes, with the compound inducing SDS–K + -precipitable DNA–topoisomerase complexes. This is the first report on luteolin as an inhibitor of the catalytic activity of topoisomerase I, and our results further support its therapeutic potential as a lead anti-cancer compound that poisons topoisomerases.

We have reductively alkylated deoxynojirimycin imino sugars using sodium cyanoborohydride to provide an efficient means of generating a series of N-alkylated compounds containing 4–18 carbon side chains. The yields were greater than 90% using a variety of aldehydes of different chain lengths, and after purification were >95% pure using 1 H-NMR. Radiolabelled compounds were prepared using sodium cyanoborotriti-ide to selectively label the first carbon atom in the alkyl chain and used in protein-binding and cell- and tissue-uptake experiments. Protein binding was chain-length-dependent with compounds of intermediate chain length (C 9 —C 12 ), demonstrating an equal distribution between the aqueous and protein-bound phase. The extent of cell uptake also increased proportionally with increased chain length in a time-dependent manner. When administered to mice, the longer alkyl-chain compounds showed reduced absorption from the intestine and a marked deposition of compound in the liver and brain, suggesting that the more hydrophobic compounds were poorly cleared by the major tissues. In tissue-culture cells compounds with 8 or fewer carbon atoms were non-toxic and had CC 50 (the concentration at which the number of cells or cell proliferation is reduced by 50%) values greater than 1mM. Compounds with chain lengths above C 8 showed a chain-length-dependent increase in cytotoxicity. N-alkylated deoxynojirimycins (C 4 —C 18 ) were evaluated for their inhibitory effects on ceramide-specific glucosyltransferase and glycoprotein-processing α-glucosidase. Increasing the alkyl chain length had little effect on α-glucosidase activity, but inhibition of ceramide-specific glucosyltransferase increased 10-fold when C 4 and C 9 —C 18 compounds were compared. Overall these data provide further definition of the molecular features of alkylated imino sugars that influence tissue selectivity and efficacy for cellular enzyme inhibition.

Multiple interactions with DNA, RNA and transcription factors occur in a transcription cycle. To survey the proximity of some of these factors to the Escherichia coli RNA polymerase surface, we produced a set of nine monoclonal antibodies (mAbs) against the enzyme. These mAbs, located at different places on the surface of the enzyme, were used in a co-immunopurification assay to investigate interference with the binding of NusA, σ70, GreB and HepA to core RNA polymerase. One of these mAbs turned out to be the first antibody inhibitor of the binding of NusA and σ70; it did not affect GreB and HepA interactions. Its epitope was located on the β′ subunit at the C-terminus of region G. The properties of this mAb reinforce the idea that the mutually exclusive binding of NusA and σ70 to core RNA polymerase is due to, at least partially, overlapping binding sites, rather than allosteric interaction between two distant binding sites. This mAb is also useful to understand the occupancy of σ70, NusA and Gre proteins on core RNA polymerase.

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, l -arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

The maize β-glucosidase isoenzymes ZMGlu1 and ZMGlu2 hydrolyse the abundant natural substrate DIMBOAGlc (2- O -β- d -glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one), whose aglycone DIMBOA (2,4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) is the major defence chemical protecting seedlings and young plant parts against herbivores and other pests. The two isoenzymes hydrolyse DIMBOAGlc with similar kinetics but differ from each other and their sorghum homologues with respect to specificity towards other substrates. To gain insights into the mechanism of substrate (i.e. aglycone) specificity between the two maize isoenzymes and their sorghum homologues, ZMGlu1 was produced in Escherichia coli , purified, crystallized and its structure solved at 2.5 Å resolution by X-ray crystallography. In addition, the complex of ZMGlu1 with the non-hydrolysable inhibitor p -nitrophenyl β- d -thioglucoside was crystallized and, based on the partial electron density, a model for the inhibitor molecule within the active site is proposed. The inhibitor is located in a slot-like active site where its aromatic aglycone is held by stacking interactions with Trp-378. Whereas some of the atoms on the non-reducing end of the glucose moiety can be modelled on the basis of the electron density, most of the inhibitor atoms are highly disordered. This is attributed to the requirement of the enzyme to accommodate two different species, namely the substrate in its ground state and in its distorted conformation, for catalysis.

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli . Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro . Using l -Leu- p -nitroanilide as the substrate, the kinetic parameters ( k cat and K m ) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

Proprotein convertase PC4A, a member of the subtilisin/kexin family of serine proteases, was obtained in enzymically active form following expression of vaccinia virus recombinant rat (r)PC4A in GH4C1 cells. It displayed maximal activity at pH 7.0 and a Ca 2+ concentration of 2.0 mM. Using PC4-specific antibodies, Western blot analysis of the medium revealed a major band at ≈ 54 kDa, corresponding to the molecular size of mature rPC4A. Among the various peptidyl-[4-methylcoumarin 7-amide (MCA)] substrates tested, the one that was preferred the most by rPC4A was acetyl (Ac)-Arg-Lys-Lys-Arg-MCA, which is cleaved 9 times faster (as judged from V max / K m measurements) than the best furin and PC1 substrate, pGlu-Arg-Thr-Lys-Arg-MCA. Recombinant rPC4A, along with human (h)furin and hPC1, cleaved a 17-amino-acid synthetic peptide, YQTL RRR V KR ↓ SLVVPTD (where ↓ denotes site of cleavage, and the important basic residues are shown in bold), encompassing the junction between the putative pro-segment of rPC4A and the active enzyme, suggesting a possible auto-activation of the enzyme. In an effort to identify potential physiological substrates for PC4, studies were performed with pro-[insulin-growth-factor (IGF)]-derived synthetic peptides, namely Ac-PA KS A R ↓ SV R A (IGF-I 66-75 ) and Ac-PA K SE R ↓ DVST (IGF-II 63-72 ), as well as two lysine mutants [(IGF-I 66-75 Lys 70 ) and (IGF-II 63-72 Lys 67 )]. Unlike PC1 and furin, rPC4A cleaved efficiently both IGF-I 66-75 and IGF-II 63-72 , suggesting a possible role of PC4 in the maturation of IGF-I and -II. In contrast, the peptides with a position 2 (P2) lysine mutation, IGF-I 66-75 Lys 70 and IGF-II 63-72 Lys 67 , were cleaved more efficiently by PC1 and furin compared with rPC4A. Furthermore, using synthetic peptides containing the processing sites of p ituitary a denylate- c yclase- a ctivating p olypeptide (PACAP)-38, we were able to confirm that, of the two testicular enzymes PC4 and PC7, PC4 is the best candidate enzyme for maturation of PACAP. Our data suggest that rPC4A is a functionally active convertase, with a substrate specificity somewhat different from that of other convertases, namely KXXR ↓ (where X denotes any other residue). As expected, p -chloromercuribenzoic acid and metal chelators such as EDTA, EGTA and trans -1,2-diaminocyclohexane- N,N,N′,N′ -tetraacetic acid inhibit the proteolytic activity of rPC4A, whereas it is activated by dithiothreitol. PC4A was also inhibited by transition-metal ions (Cu 2+ >Hg 2+ >Zn 2+ Ni 2+ >Co 2+ ), as well as by small peptide semicarbazones (SCs), such as Arg-Lys-Lys-Arg-SC ( K i 0.75 μ M) and Arg-Ser-Lys-Arg-SC ( K i 11.4 μ M).