Microbial biotransformation mechanisms of PFPiAs in soil unveiled by metagenomic analysis.

As alternatives of long-chain PFASs (Poly- and perfluoroalkyl substances), perfluoroalkyl phosphinic acids (PFPiAs) are increasingly observed in the environment, but their environmental behaviors have not been well understood. Here, the microbial biotransformation of C6/C6 and C8/C8 PFPiA in two soils (Soil N and Y) was investigated. After 252 d and 330 d of incubation with PFPiAs in Soil N and Y respectively, the levels of PFPiAs decreased distinctly, accompanied by the increasing perfluorohexaphosphonic acid (PFHxPA) or perfluorooctanophosphonic acid (PFOPA) formation, magnifying PFPiAs were susceptible to C-P cleavage, which was also confirmed by the density functional theory calculations. The half-lives of the PFPiAs were longer than one year, while generally shorter in Soil N than in Soil Y and that of C6/C6 was shorter than C8/C8 PFPiA (392 d and 746 d in Soil N, and 603 and 1155 d in Soil Y, respectively). Metagenomic sequencing analysis revealed that Proteobacteria as the primary host of the potential functional genes related to CP bond cleavage might be the crucial phyla contributing to the biotransformation of PFPiAs. Meanwhile, the more intensive interactions between the microbes in Soil N consistently contribute to its greater capacity for transforming PFPiAs.

Phosphinanes and Azaphosphinanes as Potent and Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa).

Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.

Interaction of GABAA and GABAB antagonists after status epilepticus in immature rats.

Among neurotransmitter systems affected by status epilepticus (SE) in adult rats are both GABAergic systems. To analyze possible changes of GABAA and GABAB systems in developing rats lithium-pilocarpine SE was induced at postnatal day 12 (P12). Seizures were elicited by a GABAA antagonist pentylenetetrazol (PTZ) 3, 6, 9, and 13 days after SE (i.e., in P15, P18, P21, and P25 rats), and their possible potentiation by a GABAB receptor antagonist CGP46381 was studied. Pilocarpine was replaced by saline in control animals (lithium-paraldehyde [LiPAR]). Pentylenetetrazol in a dose of 50 mg/kg s.c. elicited generalized seizures in nearly all 15-day-old naive rats and in 40% of 18-day-old ones but not in older animals. After SE, PTZ no longer elicited seizures in these two younger groups, i.e., sensitivity of GABAA system was diminished. The GABAB antagonist exhibited proconvulsant effect in P15 and P18 SE as well as LiPAR rats returning the incidence of PTZ-induced seizures to values of control animals. A decrease in the incidence of minimal clonic seizures was seen in P21 LiPAR animals; these seizures in the oldest group were not affected. Change of the effect from proconvulsant to anticonvulsant (or at least to no action) took place before postnatal day 21. Both SE and LiPAR animals exhibited similar changes but their intensity differed, effects in LiPAR controls were usually more expressed than in SE rats.

Calixarene-based phosphinic acids as inhibitors of protein tyrosine phosphatases.

In the present work, the derivatives of calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene bearing four methylene(phenyl)phosphinic acid groups on the upper rim of the macrocycle were synthesized and studied as inhibitors of human protein tyrosine phosphatases. The inhibitory capacities of the three compounds towards PTP1B were higher than those for protein tyrosine phosphatases TC-PTP, MEG1, MEG2, and SHP2. The most potent sulfonylcalix[4]arene phosphinic acid displayed Ki value of 32 nM. The thiacalix[4]arene phosphinic acid was found to be a low micromolar inhibitor of PTP1B with selectivity over the other PTPs. The kinetic experiments showed that the inhibitors compete with the substrate for the active site of the enzyme. Molecular docking was performed to explain possible binding modes of the calixarene-based phosphinic inhibitors of PTP1B.

A Phosphinate-Containing Fluorophore Capable of Selectively Inducing Apoptosis in Cancer Cells.

Chemotherapeutic agents generally suffer from off-target cytotoxicity in noncancerous cell types, leading to undesired side effects. As a result, significant effort has been put into identifying compounds that are selective for cancerous over noncancerous cell types. Our laboratory has recently developed a series of near-infrared (NIR) fluorophores containing a phosphinate functionality at the bridging position of a xanthene scaffold, termed Nebraska Red (NR) fluorophores. Herein, we report the selective cytotoxicity of one NR derivative, NR744 , against HeLa (cervical cancer) cells versus NIH-3T3 (noncancerous fibroblast) cells. Mechanistic studies based on the NIR fluorescence signal of NR744 showed distinct subcellular localization in HeLa (mitochondrial) versus NIH-3T3 (lysosomal) that resulted from the elevated mitochondrial potential in HeLa cells. This study provides a new, NIR scaffold for the further development of reagents for targeted cancer therapy.

Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes.

Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.

PcxL and HpxL are flavin-dependent, oxime-forming N-oxidases in phosphonocystoximic acid biosynthesis in Streptomyces.

Several oxime-containing small molecules have useful properties, including antimicrobial, insecticidal, anticancer, and immunosuppressive activities. Phosphonocystoximate and its hydroxylated congener, hydroxyphosphonocystoximate, are recently discovered oxime-containing natural products produced by Streptomyces sp. NRRL S-481 and Streptomyces regensis NRRL WC-3744, respectively. The biosynthetic pathways for these two compounds are proposed to diverge at an early step in which 2-aminoethylphosphonate (2AEPn) is converted to (S)-1-hydroxy-2-aminoethylphosphonate ((S)-1H2AEPn) in S. regensis but not in Streptomyces sp. NRRL S-481). Subsequent installation of the oxime moiety into either 2AEPn or (S)-1H2AEPn is predicted to be catalyzed by PcxL or HpxL from Streptomyces sp. NRRL S-481 and S. regensis NRRL WC-3744, respectively, whose sequence and predicted structural characteristics suggest they are unusual N-oxidases. Here, we show that recombinant PcxL and HpxL catalyze the FAD- and NADPH-dependent oxidation of 2AEPn and 1H2AEPn, producing a mixture of the respective aldoximes and nitrosylated phosphonic acid products. Measurements of catalytic efficiency indicated that PcxL has almost an equal preference for 2AEPn and (R)-1H2AEPn. 2AEPn was turned over at a 10-fold higher rate than (R)-1H2AEPn under saturating conditions, resulting in a similar but slightly lower kcat/Km We observed that (S)-1H2AEPn is a relatively poor substrate for PcxL but is clearly the preferred substrate for HpxL, consistent with the proposed biosynthetic pathway in S. regensis. HpxL also used both 2AEPn and (R)-1H2AEPn, with the latter inhibiting HpxL at high concentrations. Bioinformatic analysis indicated that PcxL and HpxL are members of a new class of oxime-forming N-oxidases that are broadly dispersed among bacteria.

Biological Cleavage of the C­P Bond in Perfluoroalkyl Phosphinic Acids in Male Sprague-Dawley Rats and the Formation of Persistent and Reactive Metabolites.

BACKGROUND: Perfluoroalkyl phosphinic acids (PFPiAs) have been detected in humans, wildlife, and various environmental matrices. These compounds have been used with perfluoroalkyl phosphonic acids (PFPAs) as surfactants in consumer products and as nonfoaming additives in pesticide formulations. Unlike the structurally related perfluoroalkyl sulfonic and carboxylic acids, little is known about the biological fate of PFPiAs. OBJECTIVES: We determined the biotransformation products of PFPiAs and some pharmacokinetic parameters in a rat model. METHODS: Male Sprague-Dawley rats received an oral gavage dose of either C6/C8PFPiA, C8/C8PFPiA, or C8PFPA. Blood was sampled over time, and livers were harvested upon sacrifice. Analytes were quantified using ultra-high-performance liquid chromatography-tandem mass spectrometry or gas chromatography-mass spectrometry. RESULTS: PFPiAs were metabolized to the corresponding PFPAs and 1H-perfluoroalkanes (1H-PFAs), with 70% and 75% biotransformation 2 wk after a single bolus dose for C6/C8PFPiA and C8/C8PFPiA, respectively. This is the first reported cleavage of a C-P bond in mammals, and the first attempt, with a single-dose exposure, to characterize the degradation of any perfluoroalkyl acid. Elimination half-lives were 1.9±0.5 and 2.8±0.8 days for C6/C8PFPiA and C8/C8PFPiA, respectively, and 0.95±0.17 days for C8PFPA. Although elimination half-lives were not determined for 1H-PFAs, concentrations were higher than the corresponding PFPAs 48 h after rats were dosed with PFPiAs, suggestive of slower elimination. CONCLUSIONS: PFPiAs were metabolized in Sprague-Dawley rats to form persistent PFPAs as well as 1H-PFAs, which contain a labile hydrogen that may undergo further metabolism. These results in rats produced preliminary findings of the pharmacokinetics and metabolism of PFPiAs, which should be further investigated in humans. If there is a parallel between the disposition of these chemicals in humans and rats, then humans with detectable amounts of PFPiAs in their blood may be undergoing continuous exposure. https://doi.org/10.1289/EHP1841.

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters.

Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.

The influence of angiotensin converting enzyme mutations on the kinetics and dynamics of N-domain selective inhibition.

Angiotensin-1-converting enzyme (ACE) is a zinc metalloprotease that plays a major role in blood pressure regulation via the renin-angiotensin-aldosterone system. ACE consists of two domains with differences in inhibitor binding affinities despite their 90% active site identity. While the C-domain primarily controls blood pressure, the N-domain is selective for cleavage of the antifibrotic N-acetyl-Ser-Asp-Lys-Pro. Inhibitors, such as 33RE, that selectively bind to the N-domain thus show potential for treating fibrosis without affecting blood pressure. The aim of this study was to elucidate the molecular mechanism of this selectivity. ACE inhibition by 33RE was characterized using a continuous kinetic assay with fluorogenic substrate. The N-domain displayed nanomolar (Ki = 11.21 ± 0.74 nm) and the C-domain micromolar (Ki = 11 278 ± 410 nm) inhibition, thus 1000-fold selectivity. Residues predicted to contribute to selectivity based on the N-domain-33RE co-crystal structure were subsequently mutated to their C-domain counterparts. S2 subsite mutation with resulting loss of a hydrogen bond drastically decreased the affinity (Ki = 2 794 ± 156 nm), yet did not entirely account for selectivity. Additional substitution of all unique S2 ' residues, however, completely abolished selectivity (Ki = 10 009 ± 157 nm). Interestingly, these residues do not directly bind 33RE. All mutants were therefore subjected to molecular dynamics simulations in the presence and absence of 33RE. Trajectory analyses highlighted the importance of these S2 ' residues in formation of a favourable interface between the ACE subdomains and thus a closed, ligand-bound complex. This study provides a molecular basis for the intersubsite synergism governing 33RE's 1000-fold N-selectivity and aids the future design of novel inhibitors for fibrosis treatment. ENZYMES: Angiotensin converting enzyme (ACE, EC 3.4.15.1).

Discovery of potent and selective inhibitors of human aminopeptidases ERAP1 and ERAP2 by screening libraries of phosphorus-containing amino acid and dipeptide analogues.

A collection of fifty phosphonic and phosphinic acids was screened for inhibition of ERAP1 and ERAP2, the human endoplasmic reticulum aminopeptidases. The cooperative action of these enzymes is manifested by trimming a variety of antigenic precursors to be presented on the cell surface by major histocompatibility class I. The SAR studies revealed several potent compounds, particularly among the phosphinic dipeptide analogues, that were strong inhibitors of ERAP2 (Ki=100-350nM). A wide structural diversity of the applied organophosphorus compounds, predominantly non-proteinogenic analogues, allowed identification of representatives selective toward only one form of ERAP. For example, N'-substituted α,ß-diaminophosphonates and phosphinates exhibited potency only toward ERAP2, which is in agreement with the P1 basic substrate-oriented specificity. Such discriminating ligands are invaluable tools for elucidating the precise role of a particular aminopeptidase in the concerted function of antigen processing and in human diseases.

Bioavailability and bioconcentration potential of perfluoroalkyl-phosphinic and -phosphonic acids in zebrafish (Danio rerio): Comparison to perfluorocarboxylates and perfluorosulfonates.

Currently, information regarding bioavailability and bioconcentration potential of perfluoroalkyl phosphinic acids (PFPiAs) in aquatic organisms does not exist. The main objective of the present study was to assess uptake and elimination kinetics of PFPiAs in zebrafish (Danio rerio) following aqueous exposure. The results showed that PFPiA exposure can result in very high steady-state bioconentration factors (BCFss), compared to perfluorocarboxylates and perfluorosulfonates.C6/C10 PFPiA exhibited the highest BCFss, ranging between 10(7) and 10(10), orders of magnitude higher than those for long-chain perfluorocarboxylates. Strong positive relationships were observed between BCFss versus the membrane-water distribution coefficient (Dmw) and the protein-water partition coefficient (Kpw) of the studied perfluoroalkyl substances. However, BCFss exhibited a substantial drop for the very hydrophobic PFPiAs (C8/C10 and C6/C12 PFPiAs). The reduced BCFss of these long-chain PFPiAs (perfluoroalkyl chain length=18; Dmw=10(9)) is likely the result of reduced bioavailability due to interaction with solute molecules/organic matter present in the water phase and/or reduced gill membrane permeability. While PFPiAs can be metabolized to perfluoroalkyl phosphonic acids, the metabolic transformation rate seems insufficient to counteract the high degree of uptake across gill membranes. These findings help to better understand exposure pathways and bioaccumulation behavior of these important perfluorinated acids in aquatic systems.

Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester.

A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68-93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50-55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids).

Specific inhibition by synthetic analogs of pyruvate reveals that the pyruvate dehydrogenase reaction is essential for metabolism and viability of glioblastoma cells.

The pyruvate dehydrogenase complex (PDHC) and its phosphorylation are considered essential for oncotransformation, but it is unclear whether cancer cells require PDHC to be functional or silenced. We used specific inhibition of PDHC by synthetic structural analogs of pyruvate to resolve this question. With isolated and intramitochondrial PDHC, acetyl phosphinate (AcPH, KiAcPH = 0.1 µM) was a much more potent competitive inhibitor than the methyl ester of acetyl phosphonate (AcPMe, KiAcPMe = 40 µM). When preincubated with the complex, AcPH also irreversibly inactivated PDHC. Pyruvate prevented, but did not reverse the inactivation. The pyruvate analogs did not significantly inhibit other 2-oxo acid dehydrogenases. Different cell lines were exposed to the inhibitors and a membrane-permeable precursor of AcPMe, dimethyl acetyl phosphonate, which did not inhibit isolated PDHC. Using an ATP-based assay, dependence of cellular viability on the concentration of the pyruvate analogs was followed. The highest toxicity of the membrane-permeable precursor suggested that the cellular action of charged AcPH and AcPMe requires monocarboxylate transporters. The relevant cell-specific transcripts extracted from Gene Expression Omnibus database indicated that cell lines with higher expression of monocarboxylate transporters and PDHC components were more sensitive to the PDHC inhibitors. Prior to a detectable antiproliferative action, AcPH significantly changed metabolic profiles of the investigated glioblastoma cell lines. We conclude that catalytic transformation of pyruvate by pyruvate dehydrogenase is essential for the metabolism and viability of glioblastoma cell lines, although metabolic heterogeneity causes different cellular sensitivities and/or abilities to cope with PDHC inhibition.

Human 2-oxoglutarate dehydrogenase complex E1 component forms a thiamin-derived radical by aerobic oxidation of the enamine intermediate.

Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the "ThDP-enamine"/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an "off-pathway" side reaction comprising less than 1% of the "on-pathway" reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.

Systemic exposure to the metabolites of lesogaberan in humans and animals: a case study of metabolites in safety testing.

During preclinical and early phase clinical studies of drug candidates, exposure to metabolites should be monitored to determine whether safety conclusions drawn from studies in animals can be extrapolated to humans. Metabolites accounting for more than 10% of total exposure to drug-related material (DRM) in humans are of regulatory concern, and for any such metabolites, adequate exposure should be demonstrated in animals before large-scale phase 3 clinical trials are conducted. We have previously identified six metabolites, M1-M6, of the gastroesophageal reflux inhibitor lesogaberan. In this study, we measured exposure in humans, rats, and beagle dogs to lesogaberan and these metabolites. Plasma samples were taken at various time points after lesogaberan dosing in two clinical and three preclinical studies. Concentrations of lesogaberan and its metabolites were measured, and exposures during a single dosing interval were calculated. The parent compound and metabolites M1, M2, M4, and M5 were together shown to constitute all significant exposure to DRM in humans. Only M4 and M5 were present at levels of regulatory concern (10.6% and 18.9% of total exposure to DRM, respectively, at steady state). Absolute exposure to M5 was greater in rats during toxicology studies than the highest absolute exposure observed in humans at steady state (117.0 µmol × h/liter vs. 52.2 µmol × h/liter). In contrast, exposure to M4 in rats was less than 50% of the highest absolute exposure observed in humans. Further safety testing of this metabolite may therefore be required.

Development of analytical methods for the quantification of metabolites of lesogaberan in a MIST investigation.

Analytical methods were developed for the determination of six metabolites of lesogaberan to be used in quantitative determinations of metabolites according to the guidelines of Metabolites in Safety Testing. The γ-amino butyric acid type B receptor agonist lesogaberan and its metabolites are small polar molecules and hydrophilic interaction liquid chromatography was found to be a suitable separation mode. The samples were prepared using protein precipitation and negative electrospray ionization tandem mass spectrometry was used for detection. Initially, exploratory methods for six metabolites were set up for analysis of human plasma samples taken after repeated administration of a high oral dose of lesogaberan. The purpose was to establish which metabolites were present at concentrations significant for further investigation. Four of the six metabolites were then found at clearly detectable concentrations. The analytical methods for these four metabolites were further elaborated and then taken through a qualification procedure, which showed acceptable accuracy (86-114%), precision (<9%) and good linearity in the range 0.03-5 µmol/L. No interferences were seen from endogenous plasma components.

Crystal structures of highly specific phosphinic tripeptide enantiomers in complex with the angiotensin-I converting enzyme.

Human somatic angiotensin-I converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase and a central component of the renin angiotensin aldosterone system (RAAS). Its involvement in the modulation of physiological actions of peptide hormones has positioned ACE as an important therapeutic target for the treatment of hypertension and cardiovascular disorders. Here, we report the crystal structures of the two catalytic domains of human ACE (N- and C-) in complex with FI, the S enantiomer of the phosphinic ACE/ECE-1 (endothelin converting enzyme) dual inhibitor FII, to a resolution of 1.91 and 1.85 Å, respectively. In addition, we have determined the structure of AnCE (an ACE homologue from Drosophila melanogaster) in complex with both isomers. The inhibitor FI (S configuration) can adapt to the active site of ACE catalytic domains and shows key differences in its binding mechanism mostly through the reorientation of the isoxazole phenyl side group at the P1' position compared with FII (R configuration). Differences in binding are also observed between FI and FII in complex with AnCE. Thus, the new structures of the ACE-inhibitor complexes presented here provide useful information for further exploration of ACE inhibitor pharmacophores involving phosphinic peptides and illustrate the role of chirality in enhancing drug specificity.

Phosphinic acid-based inhibitors of tubulin polyglutamylases.

Tubulin is subject to a reversible post-translational modification involving polyglutamylation and deglutamylation of glutamate residues in its C-terminal tail. This process plays key roles in regulating the function of microtubule associated proteins, neuronal development, and metastatic progression. This study describes the synthesis and testing of three phosphinic acid-based inhibitors that have been designed to inhibit both the glutamylating and deglutamylating enzymes. The compounds were tested against the polyglutamylase TTLL7 using tail peptides as substrates (100 µM) and the most potent inhibitor displayed an IC50 value of 150 µM. The incorporation of these compounds into tubulin C-terminal tail peptides may lead to more potent TTLL inhibitors.

Identification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatography.

1. In this study, hydrophilic interaction liquid chromatography (HILIC), radiochemical activity monitoring and linear trap quadrupole orbitrap mass spectrometry (MS) and tandem mass spectrometry (MS/MS) were used to identify the metabolites of a highly polar novel γ-aminobutyric acid type-B receptor agonist, lesogaberan, in rats. 2. Urine was collected from three male Wistar rats for 24 h after dosing with (14)C-labelled lesogaberan (170 mg/kg, 10 MBq/kg); plasma samples were taken 2 and 24 h after dosing. Pooled samples were separated by HILIC and eluents were analysed by radiochemical activity monitoring, MS and MS/MS. 3. Only the parent compound was detected in plasma, but six metabolites (M1-M6) were detected in urine. Analysis of MS and MS/MS data and comparison with synthetic reference standards enabled the identification of the structure of each metabolite. M1 was identified as the N-acetylated species [(2R)-3-acetamido-2-fluoropropyl]-phosphinic acid, and M6 as [(2R)-3-amino-2-fluoropropyl]-phosphonic acid. Metabolites M2 and M5 were the alcohol and carboxylic acid species 3-hydroxypropyl-phosphinic acid and 3-hydroxyphosphonoyl-propanoic acid, respectively, both of which had lost the fluorine atom present in the parent compound. M3 was the corresponding carboxylic acid species retaining the fluorine atom, (2R)-2-fluoro-3-hydroxyphosphonoyl-propanoic acid. Finally M4 was identified as [(2R)-2-fluoro-3-guanidino-propyl]-phosphinic acid.