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All Enterobacteriaceae express a polysaccharide known as enterobacterial common antigen (ECA), which is an attractive target for the development of universally acting immunotherapies. The first chemical synthesis of ECA-derived oligosaccharides for the development of such therapies is described. A number of synthetic challenges had to be addressed, including the development of concise synthetic procedures for unusual monosaccharides, the selection of appropriate orthogonal protecting groups, the development of stereoselective glycosylation methods, appropriate timing for the introduction of the carboxylic acid groups on the ManpNAcA moieties, and the selection of appropriate conditions for the reduction of multiple azido moieties. The synthetic compounds were employed to uncover immunodominant moieties of ECA. Furthermore, a monoclonal antibody (mAb) was developed that binds to ECA and can selectively recognize a wide range of Enterobacteriaceae species.

Summary: No effective targeted therapy is currently available for NRAS mutant melanoma. Experimental MEK inhibition is rather toxic and has only limited efficacy in clinical trials. At least in part, this is caused by the emergence of drug resistance, which is commonly seen for single agent treatment and shortens clinical responses. Therefore, there is a dire need to identify effective companion drug targets for NRAS mutant melanoma. Here, we show that at concentrations where single drugs had little effect, ROCK inhibitors GSK269962A or Fasudil, in combination with either MEK inhibitor GSK1120212 (Trametinib) or ERK inhibitor SCH772984 cooperatively caused proliferation inhibition and cell death in vitro. Simultaneous inhibition of MEK and ROCK caused induction of BimEL, PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.

Since the discovery of a flavin‐dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti‐TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure–activity relationships. ispartof: ChemMedChem vol:14 issue:6 pages:645-662 ispartof: location:Germany status: published

A series of 5-substituted analogs of 6-aza-2'-deoxyuridine 5'-monophosphate, 6-aza-dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-dTMP (1b), which still was recognized as a substrate by TMPKmt. ispartof: Chemistry & Biodiversity vol:9 issue:3 pages:536-56 ispartof: location:Switzerland status: published

BACKGROUND: Post-transplant anaemia and reduced muscle mass and strength are highly prevalent in kidney transplant recipients (KTRs). Decreased haemoglobin levels, a marker of anaemia, could adversely affect muscle mass and strength through multiple mechanisms, among others, through diminished tissue oxygenation. We aimed to investigate the association between haemoglobin levels with muscle mass and strength in KTRs.METHODS: We included stable KTRs from the TransplantLines Biobank and Cohort study with a functional graft ≥1 year post-transplantation. Muscle mass was assessed using 24 h urinary creatinine excretion rate (CER) and bioelectrical impedance analysis (BIA). Muscle strength was assessed with a handgrip strength test using a dynamometer and, in a subgroup (n = 290), with the five-times sit-to-stand (FTSTS) test. We used multivariable linear and logistic regression analyses to investigate the associations of haemoglobin levels with muscle mass and strength.RESULTS: In 871 included KTRs [median age 58 (interquartile range (IQR), 48-66)] years; 60% men; eGFR 51 ± 18 mL/min/1.73 m2 ) who were 3.5 (1.0-10.2) years post-transplantation, the mean serum haemoglobin level was 13.9 ± 1.8 g/dL in men and 12.8 ± 1.5 g/dL in women. Lower haemoglobin levels were independently associated with a lower CER (std. β = 0.07, P = 0.01), BIA-derived skeletal muscle mass (std. β = 0.22, P < 0.001), handgrip strength (std. β = 0.15, P < 0.001), and worse FTSTS test scores (std. β = -0.17, P = 0.02). KTRs in the lowest age-specific and sex-specific quartile of haemoglobin levels had an increased risk of being in the worst age-specific and sex-specific quartile of CER (fully adjusted OR, 2.09; 95% CI 1.15-3.77; P = 0.02), handgrip strength (fully adjusted OR, 3.30; 95% CI 1.95-5.59; P < 0.001), and FTSTS test score (fully adjusted OR, 7.21; 95% CI 2.59-20.05; P < 0.001).CONCLUSIONS: Low haemoglobin levels are strongly associated with decreased muscle mass and strength in KTRs. Future investigation will need to investigate whether maintaining higher haemoglobin levels may improve muscle mass and strength in KTRs.

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and ‐esters from diverse building blocks suitable for mmol scale synthesis on 96‐well format and on a high‐throughput nanoscale format in a highly automated fashion. High‐throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID‐19 causing agent, SARS‐CoV‐2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects., Parallel, automated, and practical multicomponent reaction synthesis of high diversity acrylamides/ester libraries and HT protein crystallography (PX) yielded potent covalent inhibitors of the main protease of the COVID‐19 causing agent SARS‐CoV‐2. More general, integration of HT PX and HT synthesis into the “design‐make‐test‐analyze” cycle will open unprecedented horizons in medicinal chemistry.

Background Many antidepressants are substrates of P-glycoprotein, an efflux transporter in the blood-brain-barrier encoded by the ABCB1 gene. Genetic variations might influence the transport rate of antidepressants and hence their pharmacological effects. This study investigates the influence of eight polymorphisms in the ABCB1 gene on antidepressant treatment response. Method 152 patients were included from psychiatric departments of the Mental Health Research Institute in Tomsk. The difference in Hamilton-Depression-Rating-Scale (HAMD-17)-scores between baseline and week two, week two and four, and baseline and week four was used to estimate timing of improvement of depression. Associations between the ABCB1 gene-polymorphisms and reduction in HAMD-17 score were assessed using independent t-test and multiple linear regression. Results Tricyclic antidepressants were associated with a higher reduction of HAMD-17 score when compared to SSRIs. The SNP rs2235040 A-allele had a significant positive influence on the Delta HAMD-17((0 -> 2W)) score but a significant negative influence on the Delta HAMD-17((2 -> 4W)) score. The rs4148739 G-allele had a significant negative influence on the Delta HAMD-17((0 -> 2W)) score but a significant positive influence on the Delta HAMD-17((2 -> 4W)) score. The SNP rs2235015 T-allele is significant negatively related to the Delta HAMD-17((2 -> 4W)) score. Conclusion ABCB1 Genetic variations appear to affect speed but not magnitude of antidepressant drug response.

The extracellular matrix (ECM) is the tissue microenvironment that regulates the characteristics of stromal and systemic cells to control processes such as inflammation and angiogenesis. Despite ongoing anti‐inflammatory treatment, low levels of inflammation exist in the airways in asthma, which alters ECM deposition by airway smooth muscle (ASM) cells. The altered ECM causes aberrant behaviour of cells, such as endothelial cells, in the airway tissue. We therefore sought to characterize the composition and angiogenic potential of the ECM deposited by asthmatic and non‐asthmatic ASM. After 72 hours under non‐stimulated conditions, the ECM deposited by primary human asthmatic ASM cells was equal in total protein, collagen I, III and fibronectin content to that from non‐asthmatic ASM cells. Further, the matrices of non‐asthmatic and asthmatic ASM cells were equivalent in regulating the growth, activity, attachment and migration of primary human umbilical vein endothelial cells (HUVECs). Under basal conditions, asthmatic and non‐asthmatic ASM cells intrinsically deposit an ECM of equivalent composition and angiogenic potential. Previous findings indicate that dysregulation of the airway ECM is driven even by low levels of inflammatory provocation. This study suggests the need for more effective anti‐inflammatory therapies in asthma to maintain the airway ECM and regulate ECM‐mediated aberrant angiogenesis.

Five new Au-III-peptidodithiocarbamato complexes of the type [(AuBr2)-Br-III(dtc-AA(1)-AA(2)-OR] (in which AA(1)=N-methylglycine (Sar), l/d-Pro; AA(2)=l/d-Ala, -aminoisobutyric acid (Aib); R=OtBu, triethylene glycol methyl ether), differing with regard to the amino acid sequence and/or the chiral amino acid configuration, were designed to enhance tumor selectivity and bioavailability. The gold(III)-based moiety was functionalized to exploit the targeting properties of the peptidomimetic ligand toward two peptide transporters (namely PEPT1 and PEPT2), which are upregulated in several tumor cells. The compounds were synthesized and fully characterized, mainly by means of elemental analysis, one- and two-dimensional NMR spectroscopy, FT-IR, and UV/Vis spectrophotometry. The crystal structures of three compounds were also solved by X-ray diffraction. In vitro cytotoxicity studies using a panel of human tumor cell lines (A549 [non-small-cell lung carcinoma], MCF-7 [breast cancer], A2780 [ovarian carcinoma], H1975 [non-small-cell lung carcinoma], H460 [large-cell lung carcinoma], and A431 [human epidermoid carcinoma]) showed the dtc-Pro-Aib-OtBu derivative to be very effective, with GI(50) values much lower than those of cisplatin. This complex was thus selected for evaluating stability under physiological conditions and possible interactions with serum albumin, as well in PARP-1 enzyme inhibition assays and preliminary ex vivo toxicity experiments on healthy rat tissues.

Ionic diode phenomena occur at asymmetric ionomer | aqueous electrolyte microhole interfaces. Depending on the applied potential, either an “open” or a “closed” diode state is observed switching between a high ion flow rate and a low ion flow rate. Physically, the “open” state is associated mainly with conductivity towards the microhole within the ionomer layer and the “closed” state is dominated by restricted diffusion-migration access to the microhole interface opposite to the ionomer. In this report we explore a “heterojunction” based on an asymmetric polymer of intrinsic microporosity (PIM) | Nafion ionomer microhole interface. Improved diode characteristics and current rectification are observed in aqueous NaCl. The effects of creating the PIM | Nafion micro-interface are investigated and suggested to lead to novel sensor architectures.

The enzyme 4‐oxalocrotonate tautomerase (4‐OT) from Pseudomonas putida mt‐2 takes part in a catabolic pathway for aromatic hydrocarbons, where it catalyzes the conversion of 2hydroxyhexa‐2,4‐dienedioate into 2‐oxohexa‐3‐enedioate. This tautomerase can also promiscuously catalyze carbon–carbon bond‐forming reactions, including various types of aldol reactions, by using its amino‐terminal proline as a key catalytic residue. Here, we used systematic mutagenesis to identify two hotspots in 4‐OT (Met45 and Phe50) at which single mutations give marked improvements in aldolase activity for the self‐condensation of propanal. Activity screening of a focused library in which these two hotspots were varied led to the discovery of a 4‐OT variant (M45Y/F50V) with strongly enhanced aldolase activity in the self‐condensation of linear aliphatic aldehydes, such as acetaldehyde, propanal, and butanal, to yield α,β‐unsaturated aldehydes. With both propanal and benzaldehyde, this double mutant, unlike the previously constructed single mutant F50A, mainly catalyzes the self‐condensation of propanal rather than the cross‐condensation of propanal and benzaldehyde, thus indicating that it indeed has altered substrate specificity. This variant could serve as a template to create new biocatalysts that lack dehydration activity and possess further enhanced aldolase activity, thus enabling the efficient enzymatic self‐coupling of aliphatic aldehydes.

The lack of selectivity for cancer cells and the resulting\ud negative impact on healthy tissue is a severe drawback of actual\ud cancer chemotherapy. Tethering of cytotoxic drugs to targeting\ud vectors such as peptides, which recognize receptors overexpressed\ud on the surface of tumor cells, is one possible strategy to overcome\ud such a problem. The pentapeptide cyc(RGDfK) targets the integrin\ud receptor αvβ3, important for tumor growth and metastasis formation.\ud In this work, two terpyridine based Ru(II) complexes were prepared\ud and for the first time conjugated to cyc(RGDfK) via amide bond\ud formation resulting in a monomeric and a dimeric bioconjugate. Both\ud Ru(II) complexes bind strongly and selectively to integrin αvβ3, with\ud the dimeric molecule displaying a 20-fold higher affinity to the\ud receptor than the monomeric one. However, the cytotoxicity of the\ud complexes and related bioconjugates against human A549 and\ud SKOV-3 cell lines is still not sufficient for application as anticancer\ud agents. Nevertheless, considering the high selectivity for integrin\ud receptor αvβ3, the synthesis of Ru-based bioconjugates with\ud cyc(RGDfK) paves a promising way towards the design of effective\ud targeted anticancer agents.

The appearance of multi‐drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross‐reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging—particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.

The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a >5000-fold improvement in catalytic efficiency (kcat /Km ) and a >10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.

A microwave-accelerated, simple, and efficient method for the construction of the 1,5-tetrazole scaffold was developed. It comprises a multicomponent reaction of an amine, a carboxylic acid derivative, and an azide source. On the basis of the availability of the archetypical starting materials, this method provided very versatile synthetic access to 1,5-disubstituted tetrazoles. The usefulness of this method was demonstrated in the synthesis of biologically important fused tetrazole scaffolds and the marketed drug cilostazol.

Background Moderate alcohol consumption has been associated with a lower risk of cardiovascular disease (CVD) and all‐cause mortality compared with heavy drinkers and abstainers. To date, studies have relied on self‐reported consumption, which may be prone to misclassification. Urinary ethyl glucuronide (EtG) is an alcohol metabolite and validated biomarker for recent alcohol consumption. We aimed to examine and compare the associations of self‐reported alcohol consumption and EtG with CVD and all‐cause mortality. Methods and Results In 5676 participants of the PREVEND (Prevention of Renal and Vascular End‐Stage Disease) study cohort, EtG was measured in 24‐hour urine samples and alcohol consumption questionnaires were administered. Participants were followed up for occurrence of first CVD and all‐cause mortality. Cox proportional hazards regression models, adjusted for age, sex, and CVD risk factors, were fitted for self‐reported consumption, divided into 5 categories: abstention, 1 to 4 units/month (reference), 2 to 7 units/week, 1 to 3 units/day, and ≥4 units/day. Similar models were fitted for EtG, analyzed as both continuous and categorical variables. Follow‐up times differed for CVD (8 years; 385 CVD events) and all‐cause mortality (14 years; 724 deaths). For both self‐reported alcohol consumption and EtG, nonsignificant trends were found toward J‐shaped associations between alcohol consumption and CVD, with higher risk in the lowest (hazard ratio for abstention versus 1–4 units/month, 1.42; 95% CI, 1.02–1.98) and highest drinking categories (hazard ratio for ≥4 units/day versus 1–4 units/month, 1.11; 95% CI, 0.68–1.84). Neither self‐report nor EtG was associated with all‐cause mortality. Conclusions Comparable associations with CVD events and all‐cause mortality were found for self‐report and EtG. This argues for the validity of self‐reported alcohol consumption in epidemiologic research.

Much effort within the nanosafety field is currently focused on the use of advanced in vitro models to reduce the gap between in vitro and in vivo studies. Within this context, precision-cut tissue slices are a unique ex vivo model to investigate nanoparticle impact using live tissue from laboratory animals and even humans. However, several aspects of the basic mechanisms of nanoparticle interactions with tissue have not yet been elucidated. To this end, liver slices are exposed to carboxylated and amino-modified polystyrene known to have a different impact on cells. As observed in standard cell cultures, amino-modified polystyrene nanoparticles induce apoptosis, and their impact is affected by the corona forming on their surface in biological fluids. Subsequently, a detailed time-resolved study of nanoparticle uptake and distribution in the tissue is performed, combining fluorescence imaging and flow cytometry on cells recovered after tissue digestion. As observed in vivo, the Kupffer cells accumulate high nanoparticle amounts and, interestingly, they move within the tissue towards the slice borders. Similar observations are reproduced in liver slices from human tissue. Thus, tissue slices can be used to reproduce ex vivo important features of nanoparticle outcomes in the liver and study nanoparticle impact on real tissue.

Glutamate is an important signaling molecule in the nervous system and its extracellular levels are regulated by amino acid transporters. Studies on the role of glutamate transport have benefitted from the development of small molecule inhibitors. Most inhibitors, however, cannot be remotely controlled with respect to the time and place of their action, which limits their application in biological studies. Herein, the development and evaluation of inhibitors of the prokaryotic transporter GltTk with photo‐controlled activity, enabling the remote, reversible, and spatiotemporally resolved regulation of transport is reported. Based on a known inhibitor, seven inhibitors, bearing a photoswitchable azobenzene moiety, are designed and synthesized. The most promising photo‐controlled inhibitor, shows in its non‐irradiated form, an IC50 of 2.5 ± 0.4 μm for transport by GltTk. Photoswitching results in a reversible drop of potency to an IC50 of 9.1 ± 1.5 μm. This 3.6‐fold difference in activity is used to demonstrate that the transporter function can be switched on and off reversibly through irradiation. As a result, this inhibitor could be a powerful tool in studying the role of glutamate transport by precisely controlling the time, and the specific tissue or groups of cells, in which the inhibitor is active.

Cancer treatment suffers from limitations that have a major impact on the patient's quality of life and survival. In the case of chemotherapy, the systemic distribution of cytotoxic drugs reduces their efficacy and causes severe side effects due to nonselective toxicity. Photopharmacology allows a novel approach to address these problems because it employs external, local activation of chemotherapeutic agents by using light. The development of photoswitchable histone deacetylase (HDAC) inhibitors as potential antitumor agents is reported herein. Analogues of the clinically used chemotherapeutic agents vorinostat, panobinostat, and belinostat were designed with a photoswitchable azobenzene moiety incorporated into their structure. The most promising compound exhibits high inhibitory potency in the thermodynamically less stable cis form and a significantly lower activity for the trans form, both in terms of HDAC activity and proliferation of HeLa cells. This approach offers a clear prospect towards local photoactivation of HDAC inhibition to avoid severe side effects in chemotherapy.

The rational design of allosteric kinase modulators is challenging but rewarding. The protein kinase PDK1, which lies at the center of the growth-factor signaling pathway, possesses an allosteric regulatory site previously validated both in vitro and in cells. ANCHOR.QUERY software was used to discover a potent allosteric PDK1 kinase modulator. Using a recently published PDK1 compound as a template, several new scaffolds that bind to the allosteric target site were generated and one example was validated. The inhibitor can be synthesized in one step by multicomponent reaction (MCR) chemistry when using the ANCHOR.QUERY approach. Our results are significant because the outlined approach allows rapid and efficient scaffold hopping from known molecules into new easily accessible and biologically active ones. Based on increasing interest in allosteric-site drug discovery, we foresee many potential applications for this approach.

Background and Objectives: The TRIP national hemovigilance and biovigilance office receives reports on side-effects and incidents associated with transfusion of labile blood products. Anaphylactic reactions accounted for the largest number of serious transfusion reactions in the period 2008-2012. In most cases, no cause is found for these reactions. TRIP data show that anaphylactic reactions occur relatively frequently with transfusion of plasma or platelet concentrates. Data from blood services show that 10% or more of plasma donors regularly use medication which is permitted under donation guidelines. It is conceivable that medication taken by the donor in plasma for transfusion could cause an anaphylactic transfusion reaction in the recipient. This exploratory study investigated the presence of drugs or drug metabolites in donor plasma. Materials and Methods: Samples (5 ml) were taken from thawed, quarantine fresh frozen plasma units (FFP) which had to be rejected for transfusion because of leaks or length of time after thawing. The samples were analysed for approximately 1000 drugs and drug metabolites using a toxicological screening method. Results: Eighty-seven samples were analysed. Toxicological screening was positive in fourteen samples (16%). In eleven samples, one substance was found, and in three samples, the presence of two or three drugs was detected. Conclusion: After freezing, storage and thawing of fresh FFP, it is possible to detect medication taken by the donor. Further investigation is recommended to analyse whether donors' medication in plasma can be implicated in some cases of allergic or anaphylactic reactions in transfusion recipients.

Enzymatic amino acid synthesis: Kinetic resolution and asymmetric synthesis of various valuable 3-substituted aspartic acids, which were obtained in fair to good yields with diastereomeric ratio values of up to98:2 and enantiomeric excess values of up to99 %, by using engineered methylaspartate ammonia lyases are described. These biocatalytic methodologies for the selective preparation of aspartic acid derivatives appear to be attractive alternatives for existing chemical methods.

Exploiting catalytic promiscuity: The proline-based enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes asymmetric Michael-type additions of linear aldehydes--ranging from acetaldehyde to octanal--to trans-β-nitrostyrene in aqueous solvent. The presence of 1.4 mol% of 4-OT effected formation of the anticipated γ-nitroaldehydes in fair to good yields with dr values of up to 93:7 and ee values of up to 81 %.

Methylaspartate ammonia-lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to yield l-threo-(2S,3S)-3-methylaspartate and l-erythro-(2S,3R)-3-methylaspartate as products. In the proposed minimal mechanism for MAL of Clostridium tetanomorphum, Lys-331 acts as the (S)-specific base catalyst and abstracts the 3S-proton from l-threo-3-methylaspartate, resulting in an enolate anion intermediate. This enolic intermediate is stabilized by coordination to the essential active site Mg2+ ion and hydrogen bonding to the Gln-329 residue. Collapse of this intermediate results in the release of ammonia and the formation of mesaconate. His-194 likely acts as the (R)-specific base catalyst and abstracts the 3R-proton from the l-erythro isomer of 3-methylaspartate, yielding the enolic intermediate. In the present study, we have investigated the importance of the residues Gln-73, Phe-170, Gln-172, Tyr-356, Thr-360, Cys-361 and Leu-384 for the catalytic activity of C. tetanomorphum MAL. These residues, which are part of the enzyme surface lining the substrate binding pocket, were subjected to site-directed mutagenesis and the mutant enzymes were characterized for their structural integrity, ability to catalyze the amination of mesaconate, and regio- and diastereoselectivity. Based on the observed properties of the mutant enzymes, combined with previous structural studies and protein engineering work, we propose a detailed catalytic mechanism for the MAL-catalyzed reaction, in which the side chains of Gln-73, Gln-172, Tyr-356, Thr-360, and Leu-384 provide favorable interactions with the substrate, which are important for substrate binding and activation. This detailed knowledge of the catalytic mechanism of MAL can serve as a guide for future protein engineering experiments., Highlights • A detailed catalytic mechanism for methylaspartate ammonia-lyase is proposed • Gln-172, Thr-360 and Cys-361 bind the 1-carboxylate group of 3-methylaspartate • Gln-73 (via a water molecule) and Gln-172 bind the 2-amino group • Tyr-356 and Leu-384 provide stabilizing interactions with the 3-methyl group

The Escherichia coli esterase YbfF displays high activity towards 1,2-O-isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R-enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2-fold higher enantioselectivity than wild-type YbfF, with an E=38 for IPG butyrate and an E=77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.

A truly 4-component reaction! In analogy to a galaxy consisting of millions of stars a multicomponent reaction scaffold can result in millions of compound variations. The MCR of α-amino acids, oxocomponents, isocyanides and primary or secondary amines is such a high-number high-diversity reaction providing an enormous potential for drug discovery or catalyst screening.

Finding the target: activity-based proteomic profiling probes based on the depalmitoylation inhibitors palmostatin B and M have been synthesized and were found to target acyl protein thioesterase 1 (APT1) and 2 (APT2) in cells.

A matter of common sense: a common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two native acyl protein thioesterase 1 (APT1) substrates. This similarity led to the design of potent inhibitors of the Ras-depalmitoylating enzyme APT1.

Most cells express a panel of different G protein-coupled receptors (GPCRs) allowing them to respond to at least a corresponding variety of extracellular ligands. In order to come to an integrative well-balanced functional response these ligand-receptor pairs can often cross-regulate each other. Although most GPCRs are fully capable to induce intracellular signalling upon agonist binding on their own, many GPCRs, if not all, appear to exist and function in homomeric and/or heteromeric assemblies for at least some time. Such heteromeric organization offers unique allosteric control of receptor pharmacology and function between the protomers and might even unmask 'new' features. However, it is important to realize that some functional consequences that are proposed to originate from heteromeric receptor interactions may also be observed due to intracellular crosstalk between signalling pathways of non-associated GPCRs. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Early detection of kinetic, metabolic, and toxicity (ADME-Tox) profiles for new drug candidates is of crucial importance during drug development. This article describes a novel in vitro system for the incubation of precision-cut liver slices (PCLS) under flow conditions, based on a poly(dimethylsiloxane) (PDMS) device containing 25-mu L microchambers for integration of the slices. The microdevice is coupled to a perifusion system, which enables a constant delivery of nutrients and oxygen and a continuous removal of waste products. Both a highly controlled incubation environment and high metabolite detection sensitivity could be achieved using microfluidics. Liver slices were viable for at least 24 h in the microdevice. The compound, 7-ethoxycoumarin (7-EC), was chosen to test metabolism, since its metabolism includes both phase I and phase 11 metabolism and when tested in the conventional well plate system, correlates well with the in vivo situation (De Kanter et al. 2004. Xenobiotica 34(3): 229-241.). The metabolic rate of 7-EC was found to be 214 +/- 5 pmol/min/mg protein in the microdevice, comparable to well plates, and was constant over time for at least 3 h. This perifusion system better mimics the in vivo situation, and has the potential to significantly contribute to drug metabolism and toxicology studies of novel chemical entities. Biotechnol. Bioeng. 2010;105: 184-194. (C) 2009 Wiley Periodicals, Inc.

H(3)R inverse agonists based on an aminopropoxy-phenyloxazoline framework constitute highly valuable druglike lead compounds that display efficacy in a mouse model of recognition memory.

Bridging the gap: The differences between medicinal chemistry at the industrial and academic levels raises the question: Is there a significant gap between the two spheres that requires attention, or should such differences be deemed natural, without the need to close the gap? Herein we provide perspectives on this issue, based in part on opinions expressed at a forum discussion held at ISMC 2008 in Vienna. (Figure Presented). © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

The identification of cell components involved in probiotic activities is a challenge in current probiotic research. In this work, a new approach based on proteomics as an analytical tool for the identification of characteristic protein profiles related to adhesion to mucin as a model probiotic property was used. Three Lactobacillus plantarum strains with different adhesion rates were used for proteomic analysis: L. plantarum WHE 92 (15.9%), L. plantarum 299 v (9.1%) and L. plantarum CECT 4185 (1.4%). Cell wall extracts were subjected to proteomic analysis of differential protein expression using 2-DE, tryptic digestion, chip-LC-QTOF mass analysis and protein identification using database search. Several proteins, previously reported to be involved in bacterial adhesion: elongation factor EF-Tu, GroEL chaperonin, molecular chaperone DnaK and glyceraldehyde-3-phosphate dehydrogenase were found to be overexpressed in the cell wall proteome of the highly adhesive strain L. plantarum WHE 92. The overexpression of two spots containing GroES co-chaperonin in the most adhesive strain also suggested the involvement of this protein in the adhesion process. The association of proteomic profiles and proteins with particular probiotic properties opens the way for the use of such profiles and proteins as bacterial biomarkers for the properties of bacteria but probably also for their potential health effects.

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5-HT) receptor subtype next to the 5-HT(1A) autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5-HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5-HT(7) receptors. Experiments were performed with the specific 5-HT(7) antagonist SB 258741 and the putative 5-HT(7) agonist AS19. In this study WAY 100.635 was used to block 5-HT(1A) receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5-HT in the ventral hippocampus as well as 5-HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co-administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5-HT in ventral hippocampus, hinting at opposed 5-HT(7) receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell-shaped dose-effect curve: moderately increasing 5-HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5-HT(7) receptors and moderate affinity for 5-HT(1A) receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5-HT neuronal activity mediated by 5-HT(7) receptors. It can be speculated, that the restoration of 5-HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5-HT(1A) receptors alone, in fact results from a shift in balance between 5-HT(1A) and 5-HT(7) receptor function.

The CXCR3 chemokine receptor was first discovered in 1996 and has been shown to play an important role in several diseases, most of which are related to inflammation. This review describes in detail the development of small CXCR3 ligands and their therapeutic potential. Classes of CXCR3 antagonists with strikingly variable core structures have emerged. Some of these compounds have confirmed the beneficial role of CXCR3 antagonism in animal models of disease. One of the compounds, AMG487, progressed to Phase II clinical trials but has been withdrawn because of lack of efficacy. New antagonist classes are being developed to reveal the full therapeutic potential of CXCR3. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of similar to 20% RSD for peak area, all sample preparation steps included, while, repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring similar to 30 times less (similar to 5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.

CXC chemokine receptor-3 (CXCR3) is a G-protein coupled receptor (GPCR) predominantly expressed on activated T lymphocytes that promote Th1 responses. Previously, we described the 3H-quinazolin-4-one containing VUF 5834 (decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]-ethyl}-(2- dimethylamino-ethyl)-amide) as a small-molecule CXCR3 antagonist with submicromolar affinity and as a lead structure for the development of CXCR3 antagonists. More recently, the related 3H-pyrido[2,3-d]pyrimidin-4-one compounds AMG 487 and NBI-74330 have been reported as nanomolar CXCR3 antagonists and these ligands are currently under clinical investigation. The aim of this study is to link the structure-activity relationship (SAR) of the previously published class of 3H-quinazolin-4-one containing CXCR3 ligands with these novel clinical candidates. From the modification of the lead structure VUF 5834 emerged the importance of the (4-fluoro-3-(trifluoromethyl)phenyl)acetyl and the 3-methylen-pyridine as substituents to improve the affinity at the human CXCR3 receptor, whereas other features are less important. The described molecules serve as tool to investigate the role of the CXCR3 receptor in various inflammatory conditions. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

Binding assays still form a fundamental part of modem drug development. Receptor binding assays are mostly based on radioactivity because of their speed, ease of use and reproducibility. Disadvantages, such as health hazards and production of radioactive waste, have prompted the development of non-radioactive receptor binding assays. This application therefore focuses on measuring receptor-ligand interactions using mass spectrometry. Moreover, the novelty of this approach originates in determining multiple analytes in a single assay (multiplexing). The proof of principle of a non-radioactive multiplex receptor assay is demonstrated using a pool of receptors from rat cortical tissue with flunitrazepam, MADAM and pindolol in one vial with or without their respective displacers. Flunitrazepam, MADAM and pindolol bound specifically at 73%, 30% and 40% to their respective receptors. This corresponds to specific binding sites of 0.61 pmol/mg protein, 0.07 pmol/mg protein and 0.06 pmol/mg protein, respectively. We propose to measure the bound fraction instead of the free fraction in order to reach a significant difference in measured signals (total binding versus non-specific binding). The bound fraction can be obtained after dissociating the ligand from the receptor-ligand complex using 50% methanol in water. The current setup of the assay calls for further improvement with respect to the measurement of binding constants for a multitude of receptors in one assay with sufficient accuracy and precision. Copyright (c) 2007 John Wiley & Sons, Ltd.

The relative stabilities of homochiral and heterochiral forms of selected dipeptides, AA, AS, AC, AV, AF, AD, AK, tripeptides, AAA, AVA, and an acetylpentapeptide, AcGLSFA, have been calculated using thermodynamic integration protocols and the GROMOS 53A6 force field. Integration pathways have been designed that produce minimal disturbance to the system, including the use of soft atoms, low-energy intermediates, and chiral inversion of the smaller amino acid in the peptide. Comparison of the results obtained by thermodynamic integration between the diastereomeric forms (in explicit water, at 300 K) and from exhaustive global minimum-energy searches for the individual dipeptides (implicit water, ε = 78, 0 K) suggests that entropic contributions to the relative stability of the chiral forms are important. This conclusion is supported by the results of explicit calculation of the effect of temperature on the relative stability of alanylvalylalanine diastereomers. The Gibbs free energy calculations predict that at ambient temperature and pressure homochiral dipeptides with small side chains or polar groups in the vicinity of the peptide backbone, AA, AS, and AD, are more stable than their heterochiral counterparts by fractions of a kJ/mol. For bigger side chains, AC, AV, AF, and AK, the heterochiral diastereomers appear to be more stable. Predicted relative stabilities are in line with observations reported in the literature for AE and YY. Excellent agreement is found for the calculated and experimentally determined relative stabilities of the diastereomers of the dipeptide AA and of all-L, AcGLSFA and its diastereomer containing D-serine in the central position. Addition of counterions to the solvent box has no significant effects on charged and neutral forms. From the present findings it would appear unlikely that the intrinsic stability difference between homo- and heterochiral dipeptides has been a driving force in a primordial selection process leading to the incorporation of amino acids with a single enantiomeric configuration in natural proteins. © 2006 Wiley Periodicals, Inc.

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.

1-[2-(4-Fluorobenzoylamino)ethyl]-4-(7-methoxynaphthyl)piperazine (S14506) is one of the most potent and selective agonists at 5-HT1A receptors. For the purpose of prospective 5-HT1A receptor imaging with positron emission tomography and the investigation of radioligand metabolic pathways, S14506 was labeled with a positron emitter, either carbon-11 (t(1/2)=20.4min) or fluorine-18 (t(1/2)=109.7min), at different positions. Thus, [O-methyl-C-11]S14506 was obtained in a radiosynthesis time of 35min by treating O-desmethyl-S14506 with [C-11]iodomethane and tetrabutylammonium hydroxide in N,N-dimethylformamide. The overall decay-corrected radiochemical yield (RCY) of [O-methyl-C-11]S14506 ranged between 6 and 24% and the specific activity (SA) between 1343 and 3101 Ci/mmol (mean 2390; n=30). [carbonyl-C-11]S14506 was synthesized through a microwave-enhanced direct coupling of in situ generated [C-11]organocarboxymagnesium bromide with amine precursor. RCYs ranged from 10 to 18%. [F-18]S14506 was prepared via nucleophilic aromatic fluoridation of the 4-nitro analog in 14 35% RCY and with SA ranging from 1063 to 2302Ci/mmol (mean 1617; n=14) in a radiosynthesis time of 115min. Heating the radiofluoridation mixture for 5min at 180 degrees C in a single mode microwave cavity gave similar RCY and SA to heating for 30 min in an oil bath at the same temperature. Copyright (c) 2005 John Wiley & Sons, Ltd.

Immunoaffinity capillary electrophoresis and surface plasmon resonance have been used for the characterization of the interaction between two large-sized proteins, the human complement protein C4 and the single-chain variable fragment C43. The rather high kinetic rate constants as determined by surface plasmon resonance pointed out that a capillary electrophoresis method had to be applied, in which the labeled C4 is preincubated with C43 before injection and the same concentration of C43 is included in the running buffer. Analysis of the concentration dependence of the small mobility shift of the fluorescent C4 signal upon binding of C43 resulted in a dissociation constant that was comparable to the one obtained with surface plasmon resonance. This study is one of the few examples where capillary electrophoresis is successfully used to characterize the interaction between large proteins.