The atypical 'hippocampal' glutamate receptor coupled to phospholipase D that controls stretch-sensitivity in primary mechanosensory nerve endings is homomeric purely metabotropic GluK2.

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings.

Chemoenzymatic Late-Stage Modifications Enable Downstream Click-Mediated Fluorescent Tagging of Peptides.

Aromatic prenyltransferases from cyanobactin biosynthetic pathways catalyse the chemoselective and regioselective intramolecular transfer of prenyl/geranyl groups from isoprene donors to an electron-rich position in these macrocyclic and linear peptides. These enzymes often demonstrate relaxed substrate specificity and are considered useful biocatalysts for structural diversification of peptides. Herein, we assess the isoprene donor specificity of the N1-tryptophan prenyltransferase AcyF from the anacyclamide A8P pathway using a library of 22 synthetic alkyl pyrophosphate analogues, of which many display reactive groups that are amenable to additional functionalization. We further used AcyF to introduce a reactive moiety into a tryptophan-containing cyclic peptide and subsequently used click chemistry to fluorescently label the enzymatically modified peptide. This chemoenzymatic strategy allows late-stage modification of peptides and is useful for many applications.

Guanidinoneomycin-maleimide molecular transporter: synthesis, chemistry and cellular uptake.

Guanidinoglycosides are a class of non-cytotoxic molecular transporters capable of delivering high molecular weight bioactive cargos into cells at low nanomolar concentrations. Efficient bioconjugation with guanidinoglycosides has been previously demonstrated by utilizing a guanidinoneomycin decorated with a reactive but also unstable N-hydroxysuccinimmide ester-containing linker. Herein we report the synthesis, chemistry, and application of a new, stable guanidinoneomycin derivative armed with a highly specific maleimide moiety which allows for thiol-maleimide click chemistry, a highly popular bioconjugation strategy, widening the field of application of these intriguing and useful delivery vehicles.

Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers.

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click" ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.

Enzymatic radiosynthesis of a 18F-Glu-Ureido-Lys ligand for the prostate-specific membrane antigen (PSMA).

Prostate cancer represents a major public health threat as it is one of the most common male cancers worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer cells in a manner that correlates with both tumour stage and clinical outcome. As such, PSMA has been identified as an attractive target for both imaging and treatment of prostate cancer. In recent years the focus on urea-based peptidomimetic inhibitors of the PSMA (representing low molecular weight/high affinity binders) has intensified as they have found use in the clinical imaging of prostate tumours. Reported herein are the design, synthesis and evaluation of a new fluorinated PSMA targeting small-molecule, FDA-PEG-GUL, which possesses the Glu-NH-CO-NH-Lys pharmacophore conjugated to a 5'-fluorodeoxy-adenosine unit. Inhibition assays were performed with FDA-PEG-GUL which revealed that it inhibits the PSMA in the nanomolar range. Additionally, it has been purposely designed so that it can be produced using the fluorinase enzyme from its chlorinated precursor, allowing for the enzymatic synthesis of radiolabelled [18F]FDA-PEG-GUL via a nucleophilic reaction that takes place in experimentally advantageous conditions (in water at neutral pH and at ambient temperature). Specific binding of [18F]FDA-PEG-GUL to PSMA expressing cancer cells was demonstrated, validating it as a promising PSMA diagnostic tool. This work establishes a successful substrate scope expansion for the fluorinase and demonstrates its first application towards targeting the PSMA.

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging.

The use of radiolabelled antibodies and antibody-derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin-avidin/streptavidin pairing, along with the inverse-electron-demand Diels-Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase-mediated transhalogenation [5'-chloro-5'-deoxyadenosine (ClDA) substrates to 5'-fluoro-5'-deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA-PEG-tetrazine and a [18 F]FDA-PEG-biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans-cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [18 F]FDA-PEG-biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase-catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C-2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [18 F]fluoride takes place in water at neutral pH and at ambient temperature.

A New Class of Fluorinated A2A Adenosine Receptor Agonist with Application to Last-Step Enzymatic [18 F]Fluorination for PET Imaging.

The A2A adenosine receptor belongs to a family of G-coupled protein receptors that have been subjected to extensive investigation over the last few decades. Due to their prominent role in the biological functions of the heart, lungs, CNS and brain, they have become a target for the treatment of illnesses ranging from cancer immunotherapy to Parkinson's disease. The imaging of such receptors by using positron emission tomography (PET) has also been of interest, potentially providing a valuable tool for analysing and diagnosing various myocardial and neurodegenerative disorders, as well as offering support to drug discovery trials. Reported herein are the design, synthesis and evaluation of two new 5'-fluorodeoxy-adenosine (FDA)-based receptor agonists (FDA-PP1 and FDA-PP2), each substituted at the C-2 position with a terminally functionalised ethynyl unit. The structures enable a synthesis of 18 F-labelled analogues by direct, last-step radiosynthesis from chlorinated precursors using the fluorinase enzyme (5'-fluoro-5'-deoxyadenosine synthase), which catalyses a transhalogenation reaction. This delivers a new class of A2A adenosine receptor agonist that can be directly radiolabelled for exploration in PET studies.

Synthesis and Superpotent Anticancer Activity of Tubulysins Carrying Non-hydrolysable N-Substituents on Tubuvaline.

Synthetic tubulysins 24 a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the α,ß-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24 a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysin A, vinorelbine and paclitaxel. Furthermore, 24 a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24 e and 24 g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24 e and 24 g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24 e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.

Synthesis, radio-synthesis and in vitro evaluation of terminally fluorinated derivatives of HU-210 and HU-211 as novel candidate PET tracers.

We report the synthesis of terminally fluorinated HU-210 and HU-211 analogues (HU-210F and HU-211F, respectively) and their biological evaluation as ligands of cannabinoid receptors (CB1 and CB2) and N-methyl d-aspartate receptor (NMDAR). [18F]-labelled HU-210F was radiosynthesised from the bromo-substituted precursor. In vitro assays showed that both HU-210F and HU-211F retain the potent pharmacological profile of HU-210 and HU-211, suggesting that [18F]-radiolabelled HU-210F and HU-211F could have potential as PET tracers for in vivo imaging.

Design, synthesis, in vitro characterization and preliminary imaging studies on fluorinated bile acid derivatives as PET tracers to study hepatic transporters.

With the aim of identifying a fluorinated bile acid derivative that could be used as [18F]-labeled Positron Emission Tomography (PET) tracer for imaging the in vivo functioning of liver transporter proteins, and particularly of OATP1B1, three fluorinated bile acid triazole derivatives of cholic, deoxycholic and lithocholic acid (CATD, DCATD and LCATD 4a-c, respectively) were synthesized and labeled with tritium. In vitro transport properties were studied with cell-based assays to identify the best substrate for OATP1B1. In addition, the lead compound, LCATD (4c), was tested as a substrate of other liver uptake transporters OATP1B3, NTCP and efflux transporter BSEP to evaluate its specificity of liver transport. The results suggest that 4c is a good substrate of OATP1B1 and NTCP, whereas it is a poor substrate of OATP1B3. The efflux transporter BSEP also appears to be involved in the excretion of 4c from hepatocytes. The automated radiosynthesis of [18F]-4c was accomplished in a multi-GBq scale and a pilot imaging experiment in a wild type rat was performed after i.v. administration to assess the biodistribution and clearance of the tracer. PET imaging revealed that radioactivity was primarily located in the liver (tmax=75s) and cleared exclusively through the bile, thus allowing to image the hepatobiliary excretion of bile acids in the animal model. These findings suggest that [18F]-LCATD 4c is a promising PET probe for the evaluation of hepatic transporters OATP1B1, NTCP and BSEP activity with potential for studying drug-drug interactions and drug-induced toxicity involving these transporters.

Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy.

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5s), therefore direct NO quantification is challenging. An indirect method - based on quantifying conversion of an l-Arg- to l-Cit-derivative by 1H NMR spectroscopy - is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a-j and 11a-c were better or equivalent substrates for the eNOS enzyme (NO2- production=19-46µM) than native l-Arg (NO2- production=25µM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by 1H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.

NGR Tumor-Homing Peptides: Structural Requirements for Effective APN (CD13) Targeting.

Cyclic CNGRC (cCNGRC) peptides are very important targeting ligands for Aminopeptidase N (APN or CD13), which is overexpressed on the surface of many cancer cells. In this work we have (1) developed an efficient solid-phase synthesis and (2) tested on purified porcine APN and APN-expressing human cells two different classes of cCNGRC peptides: the first carrying a biotin affinity tag or a fluorescent tag attached to the carboxyl Arg-Cys-COOH terminus and the second with the tags attached to the amino H2N-Cys-Asn terminus. Carboxyl-terminus functionalized cCNGRC peptides 3, 6, and 8 showed good affinity for porcine APN and very good capacity to target and be internalized into APN-expressing cells. In contrast, amino-terminus functionalized cCNGRC peptides 4, 5, and 7 displayed significantly decreased affinity and targeting capacity. These results, which are in agreement with the recently reported X-ray structure of a cCNGRC peptide bound to APN showing important stabilizing interactions between the unprotected cCNGRC amino terminus and the APN active site, indicate that the carboxyl and not the amino-terminus of cCNGRC peptides should be used as a "handle" for the attachment of toxic payloads for therapy or isotopically labeled functions for imaging and nuclear medicine.

Last-Step Enzymatic [(18) F]-Fluorination of Cysteine-Tethered RGD Peptides Using Modified Barbas Linkers.

We report a last-step fluorinase-catalyzed [(18) F]-fluorination of a cysteine-containing RGD peptide. The peptide was attached through sulfur to a modified and more hydrophilic variant of the recently disclosed Barbas linker which was itself linked to a chloroadenosine moiety via a PEGylated chain. The fluorinase was able to use this construct as a substrate for a transhalogenation reaction to generate [(18) F]-radiolabeled RGD peptides, which retained high affinity to cancer-cell relevant αv ß3 integrins.

Arginine analogues incorporating carboxylate bioisosteric functions are micromolar inhibitors of human recombinant DDAH-1.

Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme involved in the metabolism of asymmetric dimethylarginine (ADMA) and N-monomethyl arginine (NMMA), which are endogenous inhibitors of the nitric oxide synthase (NOS) family of enzymes. Two isoforms of DDAH have been identified in humans, DDAH-1 and DDAH-2. DDAH-1 inhibition represents a promising strategy to limit the overproduction of NO in pathological states without affecting the homeostatic role of this important messenger molecule. Here we describe the design and synthesis of 12 novel DDAH-1 inhibitors and report their derived kinetic parameters, IC50 and Ki. Arginine analogue 10a, characterized by an acylsulfonamide isosteric replacement of the carboxylate, showed a 13-fold greater inhibitory potential relative to the known DDAH-1 inhibitor, L-257. Compound 10a was utilized to study the putative binding interactions of human DDAH-1 inhibition using molecular dynamics simulations. The latter suggests that several stabilizing interactions occur in the DDAH-1 active-site, providing structural insights for the enhanced inhibitory potential demonstrated by in vitro inhibition studies.

Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis.

Sinorhizobium meliloti differentiates into persisting, nitrogen-fixing bacteroids within root nodules of the legume Medicago truncatula. Nodule-specific cysteine-rich antimicrobial peptides (NCR AMPs) and the bacterial BacA protein are essential for bacteroid development. However, the bacterial factors central to the NCR AMP response and the in planta role of BacA are unknown. We investigated the hypothesis that BacA is critical for the bacterial response towards NCR AMPs. We found that BacA was not essential for NCR AMPs to induce features of S. meliloti bacteroids in vitro. Instead, BacA was critical to reduce the amount of NCR AMP-induced membrane permeabilization and bacterial killing in vitro. Within M. truncatula, both wild-type and BacA-deficient mutant bacteria were challenged with NCR AMPs, but this resulted in persistence of the wild-type bacteria and rapid cell death of the mutant bacteria. In contrast, BacA was dispensable for bacterial survival in an M. truncatula dnf1 mutant defective in NCR AMP transport to the bacterial compartment. Therefore, BacA is critical for the legume symbiosis by protecting S. meliloti against the bactericidal effects of NCR AMPs. Host AMPs are ubiquitous in nature and BacA proteins are essential for other chronic host infections by symbiotic and pathogenic bacteria. Hence, our findings suggest that BacA-mediated protection of bacteria against host AMPs is a critical stage in the establishment of different prolonged host infections.

Protein tyrosine phosphatase 1B in metabolic diseases and drug development.

Protein tyrosine phosphatase 1B (PTP1B), a non-transmembrane phosphatase, has a major role in a variety of signalling pathways, including direct negative regulation of classic insulin and leptin signalling pathways, and is implicated in the pathogenesis of several cardiometabolic diseases and cancers. As such, PTP1B has been a therapeutic target for over two decades, with PTP1B inhibitors identified either from natural sources or developed throughout the years. Some of these inhibitors have reached phase I and/or II clinical trials in humans for the treatment of type 2 diabetes mellitus, obesity and/or metastatic breast cancer. In this Review, we summarize the cellular processes and regulation of PTP1B, discuss evidence from in vivo preclinical and human studies of the association between PTP1B and different disorders, and discuss outcomes of clinical trials. We outline challenges associated with the targeting of this phosphatase (which was, until the past few years, viewed as difficult to target), the current state of the field of PTP1B inhibitors (and dual phosphatase inhibitors) and future directions for manipulating the activity of this key metabolic enzyme.

Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide.

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

Efficient bioconjugation of 5-fluoro-5-deoxy-ribose (FDR) to RGD peptides for positron emission tomography (PET) imaging of α(v)ß(3) integrin receptor.

The utility of 5-fluoro-5-deoxyribose (FDR) as an efficient bioconjugation agent for radiolabelling of the RGD peptides c(RGDfK) and c(RGDfC) is demonstrated. The bioconjugation is significantly superior to that achieved with 2-fluoro-2-deoxyglucose (FDG) and benefits from the location of the fluorine at C-5, and that ribose is a 5-membered ring sugar rather than a 6-membered ring. Both features favour ring opening to the aldehydic form of the sugar to promote smooth oxime ligation with aminooxy ether functionalised peptides. [(18)F]FDR was prepared in this study by synthesis from fluoride-18 using an automated synthesis protocol adapting that used routinely for [(18)F]FDG. c(RGDfK) was functionalised with an aminooxyacetyl group (Aoa) via its lysine terminus, while c(RGDfC) was functionalised with an aminooxyhexylmaleimide (Ahm) through a cysteine-maleimide conjugation. Bioconjugation of [(18)F]FDR to c(RGDfC)-Ahm proved to be more efficient than c(RGDfK)-Aoa (92% versus 65%). The unlabelled ((19)F) bioconjugates c(RGDfK)-Aoa-FDR and c(RGDfC)-Ahm-FDR were prepared and their in vitro affinity to purified integrin αvß3 was determined. c(RGDfK)-Aoa-FDR showed the greater affinity. Purified "hot" bioconjugates c(RGDfK)-Aoa-[(18)F]FDR and c(RGDfC)-Ahm-[(18)F]FDR were assayed by incubation with MCF7, LNCaP and PC3 cell lines. In both cases the conjugated RGD peptides showed selectivity for PC3 cells, which express αvß3 integrin, with the c(RGDfK)-Aoa-[(18)F]FDR demonstrating better binding, consistent with its higher in vitro affinity. The study demonstrates that [(18)F]FDR is an efficient bioconjugation ligand for RGD bioactive peptides.

Biochemical characterization of a cyanobactin arginine-N-prenylase from the autumnalamide biosynthetic pathway.

Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-D-Arg-containing autumnalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.

Clinical value of 3'-deoxy-3'-[18F]fluorothymidine-positron emission tomography for diagnosis, staging and assessing therapy response in lung cancer.

Lung cancer has the highest mortality rate of any tumour type. The main driver of lung tumour growth and development is uncontrolled cellular proliferation. Poor patient outcomes are partly the result of the limited range of effective anti-cancer therapies available and partly due to the limited accuracy of biomarkers to report on cell proliferation rates in patients. Accordingly, accurate methods of diagnosing, staging and assessing response to therapy are crucial to improve patient outcomes. One effective way of assessing cell proliferation is to employ non-invasive evaluation using 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography [18F]FLT-PET. [18F]FLT, unlike the most commonly used PET tracer [18F]fluorodeoxyglucose ([18F]FDG), can specifically report on cell proliferation and does not accumulate in inflammatory cells. Therefore, this radiotracer could exhibit higher specificity in diagnosis and staging, along with more accurate monitoring of therapy response at early stages in the treatment cycle. This review summarises and evaluates published studies on the clinical use of [18F]FLT to diagnose, stage and assess response to therapy in lung cancer.