An Investigation into the In Vitro Metabolic Stability of Aryl Sulfonyl Fluorides for their Application in Medicinal Chemistry and Radiochemistry.

Molecules that feature a sulfonyl fluoride (SO2F) moiety have been gaining increasing interest due to their unique reactivity and potential applications in synthetic chemistry, medicinal chemistry, and other biological uses. A particular interest is towards 18F-radiochemistry where sulfonyl fluorides can be used as a method to radiolabel biomolecules or can be used as radiofluoride relay reagents that facilitate radiolabeling of other molecules. The low metabolic stability of sulfonyl fluoride S-F bonds, however, presents an issue and limits the applicability of sulfonyl fluorides. The aim of this work was to increase understanding of what features contribute to the metabolic instability of the S-F bond in model aryl sulfonyl fluorides and identify approaches to increasing sulfonyl fluoride stability for 18F-radiochemistry and other medicinal, synthetic chemistry and biological applications. To undertake this, 14 model aryl sulfonyl fluorides compounds with varying functional groups and substitution patterns were investigated, and their stabilities were examined in various media, including phosphate-buffered saline and rat serum as a model for biological conditions. The results indicate that both electronic and steric factors affect the stability of the S-F bond, with the 2,4,6-trisubstituted model aryl sulfonyl fluorides examined displaying the highest in vitro metabolic stability.

Electrophysiological Responses of Bactrocera kraussi (Hardy) (Tephritidae) to Rectal Gland Secretions and Headspace Volatiles Emitted by Conspecific Males and Females.

Pheromones are biologically important in fruit fly mating systems, and also have potential applications as attractants or mating disrupters for pest management. Bactrocera kraussi (Hardy) (Diptera: Tephritidae) is a polyphagous pest fruit fly for which the chemical profile of rectal glands is available for males but not for females. There have been no studies of the volatile emissions of either sex or of electrophysiological responses to these compounds. The present study (i) establishes the chemical profiles of rectal gland contents and volatiles emitted by both sexes of B. kraussi by gas chromatography-mass spectrometry (GC-MS) and (ii) evaluates the detection of the identified compounds by gas chromatography-electroantennogram detection (GC-EAD) and -electropalpogram detection (GC-EPD). Sixteen compounds are identified in the rectal glands of male B. kraussi and 29 compounds are identified in the rectal glands of females. Of these compounds, 5 were detected in the headspace of males and 13 were detected in the headspace of females. GC-EPD assays recorded strong signals in both sexes against (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 2-ethyl-7-mehtyl-1,6-dioxaspiro[4.5]decane isomer 2, (E,Z)/(Z,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, and (Z,Z)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. Male antennae responded to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane, 2-methyl-6-pentyl-3,4-dihydro-2H-pyran, 6-hexyl-2-methyl-3,4-dihydro-2H-pyran, 6-oxononan-1-ol, ethyl dodecanoate, ethyl tetradecanoate and ethyl (Z)-hexadec-9-enoate, whereas female antennae responded to (E,E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and 2-methyl-6-pentyl-3,4-dihydro-2H-pyran only. These compounds are candidates as pheromones mediating sexual interactions in B. kraussi.

Methods to Enhance the Metabolic Stability of Peptide-Based PET Radiopharmaceuticals.

The high affinity and specificity of peptides towards biological targets, in addition to their favorable pharmacological properties, has encouraged the development of many peptide-based pharmaceuticals, including peptide-based positron emission tomography (PET) radiopharmaceuticals. However, the poor in vivo stability of unmodified peptides against proteolysis is a major challenge that must be overcome, as it can result in an impractically short in vivo biological half-life and a subsequently poor bioavailability when used in imaging and therapeutic applications. Consequently, many biologically and pharmacologically interesting peptide-based drugs may never see application. A potential way to overcome this is using peptide analogues designed to mimic the pharmacophore of a native peptide while also containing unnatural modifications that act to maintain or improve the pharmacological properties. This review explores strategies that have been developed to increase the metabolic stability of peptide-based pharmaceuticals. It includes modifications of the C- and/or N-termini, introduction of d- or other unnatural amino acids, backbone modification, PEGylation and alkyl chain incorporation, cyclization and peptide bond substitution, and where those strategies have been, or could be, applied to PET peptide-based radiopharmaceuticals.

Ketimine reductase/CRYM catalyzes reductive alkylamination of α-keto acids, confirming its function as an imine reductase.

Recently, crystalized mouse ketimine reductase/CRYM complexed with NADPH was found to have pyruvate bound in its active site. We demonstrate that the enzyme binds α-keto acids, such as pyruvate, in solution, and catalyzes the formation of N-alkyl-amino acids from alkylamines and α-keto acids (via reduction of imine intermediates), but at concentrations of these compounds not expected to be encountered in vivo. These findings confirm that, mechanistically, ketimine reductase/CRYM acts as a classical imine reductase and may explain the finding of bound pyruvate in the crystallized protein.

Insights into Enzyme Catalysis and Thyroid Hormone Regulation of Cerebral Ketimine Reductase/µ-Crystallin Under Physiological Conditions.

Mammalian ketimine reductase is identical to µ-crystallin (CRYM)-a protein that is also an important thyroid hormone binding protein. This dual functionality implies a role for thyroid hormones in ketimine reductase regulation and also a reciprocal role for enzyme catalysis in thyroid hormone bioavailability. In this research we demonstrate potent sub-nanomolar inhibition of enzyme catalysis at neutral pH by the thyroid hormones L-thyroxine and 3,5,3'-triiodothyronine, whereas other thyroid hormone analogues were shown to be far weaker inhibitors. We also investigated (a) enzyme inhibition by the substrate analogues pyrrole-2-carboxylate, 4,5-dibromopyrrole-2-carboxylate and picolinate, and (b) enzyme catalysis at neutral pH of the cyclic ketimines S-(2-aminoethyl)-L-cysteine ketimine (owing to the complex nomenclature trivial names are used for the sulfur-containing cyclic ketimines as per the original authors' descriptions) (AECK), Δ(1)-piperideine-2-carboxylate (P2C), Δ(1)-pyrroline-2-carboxylate (Pyr2C) and Δ(2)-thiazoline-2-carboxylate. Kinetic data obtained at neutral pH suggests that ketimine reductase/CRYM plays a major role as a P2C/Pyr2C reductase and that AECK is not a major substrate at this pH. Thus, ketimine reductase is a key enzyme in the pipecolate pathway, which is the main lysine degradation pathway in the brain. In silico docking of various ligands into the active site of the X-ray structure of the enzyme suggests an unusual catalytic mechanism involving an arginine residue as a proton donor. Given the critical importance of thyroid hormones in brain function this research further expands on our knowledge of the connection between amino acid metabolism and regulation of thyroid hormone levels.

Characterisation of a novel UV filter in the lens of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus).

Structural analysis of a novel UV filter present in the lens of the thirteen-lined ground squirrel has shown that it is related in structure to N-acetyl-3-hydroxykynurenine. This finding is consistent with the fact that the squirrel lenses also contain high levels of this tryptophan metabolite. Analysis of both NMR and mass spectrometric data suggested that the novel UV filter compound forms by condensation of proline with N-acetyl-3-hydroxykynurenine. Its absorption maximum at 340 nm is more than 20 nm lower than that of the kynurenines and it may therefore assist in filtering the more damaging shorter wavelengths of UVA.

Separate mechanisms for age-related truncation and racemisation of peptide-bound serine.

Some amino acids are particularly susceptible to degradation in long-lived proteins. Foremost among these are asparagine, aspartic acid and serine. In the case of serine residues, cleavage of the peptide bond on the N-terminal side, as well as racemisation, has been observed. To investigate the role of the hydroxyl group, and whether cleavage and racemisation are linked by a common mechanism, serine peptides with a free hydroxyl group were compared to analogous peptides where the serine hydroxyl group was methylated. Peptide bond cleavage adjacent to serine was increased when the hydroxyl group was present, and this was particularly noticeable when it was present as the hydroxide ion. Adjacent amino acid residues also had a pronounced affect on cleavage at basic pH, with the SerPro motif being especially susceptible to scission. Methylation of the serine hydroxyl group abolished truncation, as did insertion of a bulky amino acid on the N-terminal side of serine. By contrast, racemisation of serine occurred to a similar extent in both O-methylated and unmodified peptides. On the basis of these data, it appears that racemisation of Ser, and cleavage adjacent to serine, occur via separate mechanisms. Addition of water across the double bond of dehydroalanine was not detected, suggesting that this mechanism was unlikely to be responsible for conversion of L-serine to D-serine. Abstraction of the alpha proton may account for the majority of racemisation of serine in proteins.

Imine reductases: a comparison of glutamate dehydrogenase to ketimine reductases in the brain.

A key intermediate in the glutamate dehydrogenase (GDH)-catalyzed reaction is an imine. Mechanistically, therefore, GDH exhibits similarities to the ketimine reductases. In the current review, we briefly discuss (a) the metabolic importance of the GDH reaction in liver and brain, (b) the mechanistic similarities between GDH and the ketimine reductases, (c) the metabolic importance of the brain ketimine reductases, and (d) the neurochemical consequences of defective ketimine reductases. Our review contains many historical references to the early work on amino acid metabolism. This work tends to be overlooked nowadays, but is crucial for a contemporary understanding of the central importance of ketimines in nitrogen and intermediary metabolism. The ketimine reductases are important enzymes linking nitrogen flow among several key amino acids, yet have been little studied. The cerebral importance of the ketimine reductases is an area of biomedical research that deserves far more attention.

Mutation of cysteine residues alters the heme-binding pocket of indoleamine 2,3-dioxygenase-1.

The hemoprotein indoleamine 2,3-dioxygenase-1 (IDO1) is the first and rate-limiting enzyme in mammalian tryptophan metabolism. Interest in IDO1 continues to grow, due to the ever expanding influence IDO1 plays in the immune response. This study examined the contribution of all individual cysteine residues towards the overall catalytic properties and stability of recombinant human IDO1 via mutagenesis studies using a range of biochemical and spectroscopic techniques, including in vitro kinetic assessment, secondary structure identification via circular dichroism spectroscopy and thermal stability assessment. Upon mutation of cysteine residues we observed changes in secondary structure (principally, shifting from α-helix/ß-sheet features to random coil structures) that produced out of plane heme torsion and puckering, changes to thermal stability (including gains in stability for one mutant protein) and differences in enzymatic activity (such as, increased ability to convert non-natural substrates, e.g.d-tryptophan) from wild type IDO1 enzyme.

Lysine metabolism in mammalian brain: an update on the importance of recent discoveries.

The lysine catabolism pathway differs in adult mammalian brain from that in extracerebral tissues. The saccharopine pathway is the predominant lysine degradative pathway in extracerebral tissues, whereas the pipecolate pathway predominates in adult brain. The two pathways converge at the level of ∆(1)-piperideine-6-carboxylate (P6C), which is in equilibrium with its open-chain aldehyde form, namely, α-aminoadipate δ-semialdehyde (AAS). A unique feature of the pipecolate pathway is the formation of the cyclic ketimine intermediate ∆(1)-piperideine-2-carboxylate (P2C) and its reduced metabolite L-pipecolate. A cerebral ketimine reductase (KR) has recently been identified that catalyzes the reduction of P2C to L-pipecolate. The discovery that this KR, which is capable of reducing not only P2C but also other cyclic imines, is identical to a previously well-described thyroid hormone-binding protein [µ-crystallin (CRYM)], may hold the key to understanding the biological relevance of the pipecolate pathway and its importance in the brain. The finding that the KR activity of CRYM is strongly inhibited by the thyroid hormone 3,5,3'-triiodothyronine (T3) has far-reaching biomedical and clinical implications. The inter-relationship between tryptophan and lysine catabolic pathways is discussed in the context of shared degradative enzymes and also potential regulation by thyroid hormones. This review traces the discoveries of enzymes involved in lysine metabolism in mammalian brain. However, there still remain unanswered questions as regards the importance of the pipecolate pathway in normal or diseased brain, including the nature of the first step in the pathway and the relationship of the pipecolate pathway to the tryptophan degradation pathway.

Discovery and characterisation of hydrazines as inhibitors of the immune suppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1).

Screening of a fragment library identified 2-hydrazinobenzothiazole as a potent inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme expressed by tumours that suppresses the immune system. Spectroscopic studies indicated that 2-hydrazinobenzothiazole interacted with the IDO1 haem and in silico docking predicted that the interaction was through hydrazine. Subsequent studies of hydrazine derivatives identified phenylhydrazine (IC50=0.25 ± 0.07 µM) to be 32-fold more potent than 2-hydrazinobenzothiazole (IC50=8.0 ± 2.3 µM) in inhibiting rhIDO1 and that it inhibited cellular IDO1 at concentrations that were noncytotoxic to cells. Here, phenylhydrazine is shown to inhibit IDO1 through binding to haem.

Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme whose expression by a broad range of clinical tumors is associated with immunosuppression and poor patient outcome. Here we describe a new fluorescence assay for measuring IDO1 activity suitable for high-throughput screening of compound libraries for novel IDO1 inhibitors. This assay is easy to perform, requiring the addition of only one reagent prior to readout. In place of measuring kynurenine, it uses the in situ formation of an N-formylkynurenine-derived fluorophore (NFKPIP) measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. The fluorescence intensity of the NFKPIP formed is directly related to the amount of enzyme activity, and the signal is stable over 8 h. This assay has a lower limit of detection, equating to 153 nM N-formylkynurenine, which is over 30-fold lower than the limits of detection of existing assays for IDO1 activity. When we compared the performance of the new assay with that of the published colorimetric absorbance assay in screening the National Cancer Institute Diversity Set III of 1,597 compounds for IDO1 inhibitors, we obtained an identical list of the 25 most active compounds in the two assays. Although 93 compounds (aldehydes, ketones, and aromatic amines) in the library interfered with the absorbance readout, only 18 compounds (conjugated systems and fused cycles) interfered with the readout of the new fluorescence assay. IC(50) values determined using the new assay for three known IDO1 inhibitors-1,4-naphthoquinone, 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide and 4-phenyl-1H-imidazole-were consistent with their literature values, further validating the new assay for measuring IDO1 activity.

Is protein methylation in the human lens a result of non-enzymatic methylation by S-adenosylmethionine?

Since crystallins in the human lens do not turnover, they are susceptible to modification by reactive molecules over time. Methylation is a major post-translational lens modification, however the source of the methyl group is not known and the extent of modification across all crystallins has yet to be determined. Sites of methylation in human lens proteins were determined using HPLC/mass spectrometry following digestion with trypsin. The overall extent of protein methylation increased with age, and there was little difference in the extent of modification between soluble and insoluble crystallins. Several different cysteine and histidine residues in crystallins from adult lenses were found to be methylated with one cysteine (Cys 110 in γD crystallin) at a level approaching 70%, however, methylation of crystallins was not detected in fetal or newborn lenses. S-adenosylmethionine (SAM) was quantified at significant (10-50 µM) levels in lenses, and in model experiments SAM reacted readily with N-α-tBoc-cysteine and N-α-tBoc-histidine, as well as ßA3-crystallin. The pattern of lens protein methylation seen in the human lens was consistent with non-enzymatic alkylation. The in vitro data shows that SAM can act directly to methylate lens proteins and SAM was present in significant concentrations in human lens. Thus, non-enzymatic methylation of crystallins by SAM offers a possible explanation for this major human lens modification.

Novel indoleamine 2,3-dioxygenase-1 inhibitors from a multistep in silico screen.

Indoleamine 2,3-dioxygenase-1 (IDO-1) is a heme containing enzyme that catalyses the initial step in the major pathway of l-tryptophan catabolism; the kynurenine pathway. A large body of evidence has been accumulating for its immunosuppressive and tumoural escape roles and its applicability as a therapeutic target. Of particular interest is the possibility that IDO-1 inhibition may arrest, and sometimes revert, tumour growth. There exists a continuing need for the development of new and specific inhibitors for IDO-1, and we have created three pharmacophores designed to aid in this search. Initial database hits were further screened using Kier flexibility and a 'What-If' docking technique, designed to overcome the inherent limitations of today's forcefields with regards to heme chemistry. Eighteen compounds were tested in vitro, yielding four novel inhibitors with low micromolar IC(50) values, comparable with current inhibitors.

Mammalian forebrain ketimine reductase identified as µ-crystallin; potential regulation by thyroid hormones.

Ketimine reductase (E.C. 1.5.1.25) was purified to apparent homogeneity from lamb forebrain by means of a rapid multi-step chromatography protocol. The purified enzyme was identified by MS/MS (mass spectrometry) as µ-crystallin. The identity was confirmed by heterologously expressing human µ-crystallin in Escherichia coli and subsequent chromatographic purification of the protein. The purified human µ-crystallin was confirmed to have ketimine reductase activity with a maximum specific activity similar to that of native ovine ketimine reductase, and was found to catalyse a sequential reaction. The enzyme substrates are putative neuromodulator/transmitters. The thyroid hormone 3,5,3'-l-triiodothyronine (T3) was found to be a strong reversible competitive inhibitor, and may have a novel role in regulating their concentrations. µ-Crystallin is also involved in intracellular T3 storage and transport. This research is the first to demonstrate an enzyme function for µ-crystallin. This newly demonstrated enzymatic activity identifies a new role for thyroid hormones in regulating mammalian amino acid metabolism, and a possible reciprocal role of enzyme activity regulating bioavailability of intracellular T3.

Mouse and human indoleamine 2,3-dioxygenase display some distinct biochemical and structural properties.

The hemoprotein indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the most significant pathway for mammalian tryptophan metabolism. It has received considerable attention in recent years, particularly due to its dual role in immunity and the pathogenesis of many diseases. Reported here are differences and similarities between biochemical behaviour and structural features of recombinant human IDO and recombinant mouse IDO. Significant differences were observed in the conversion of substrates and pH stability. Differences in inhibitor potency and thermal stability were also noted. Secondary structural features were broadly similar but variation between species was apparent, particularly in the alpha-helix portion of the enzymes. With mouse models substituting for human diseases, the differences between mouse and human IDO must be recognised before applying experimental findings from one system to the next.

Systematic Modification of Zingerone Reveals Structural Requirements for Attraction of Jarvis's Fruit Fly.

Tephritid fruit flies are amongst the most significant horticultural pests globally and male chemical lures are important for monitoring and control. Zingerone has emerged as a unique male fruit fly lure that can attract dacine fruit flies that are weakly or non-responsive to methyl eugenol and cuelure. However, the key features of zingerone that mediate this attraction are unknown. As Jarvis's fruit fly, Bactrocera jarvisi (Tryon), is strongly attracted to zingerone, we evaluated the response of B. jarvisi to 37 zingerone analogues in a series of field trials to elucidate the functional groups involved in attraction. The most attractive analogues were alkoxy derivatives, with isopropoxy being the most attractive, followed by ethoxy and trifluoromethoxy analogues. All of the phenolic esters tested were also attractive with the response typically decreasing with increasing size of the ester. Results indicate that the carbonyl group, methoxy group, and phenol of zingerone are key sites for the attraction of B. jarvisi and identify some constraints on the range of structural modifications that can be made to zingerone without compromising attraction. These findings are important for future work in developing and optimising novel male chemical lures for fruit flies.

Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage.

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.

Reversible binding of kynurenine to lens proteins: potential protection by glutathione in young lenses.

PURPOSE: Human ultraviolet light (UV) filters, such as kynurenine (Kyn), readily deaminate to reactive unsaturated ketones that covalently modify proteins in older human lenses. The aim of this study was to examine in vitro rates of formation and decomposition of the three major Kyn-amino acid adducts and possible consequences for the lens. METHODS: The t-Boc-protected Kyn-His, Kyn-Lys, and Kyn-Cys adducts and Kyn-Cys were synthesized from the corresponding amino acids and Kyn. Calf lens proteins were modified with Kyn by incubation at pH 7. Stability and competition studies of the adducts were conducted under physiological conditions. Kyn-amino acids and their decomposition products were quantified using HPLC. RESULTS: At physiological pH, Kyn-Cys adducts formed more rapidly than either Lys or His adducts, but they also decomposed readily. By contrast, His adducts were stable. Cysteine (Cys) residues in beta-crystallins were major sites of modification. The Kyn moiety, initially bound to Cys residues, was found to transfer to other amino acids. Glutathione promoted the breakdown of Kyn-Cys. CONCLUSIONS: These data may help explain why proteins in young lenses are not modified by UV filters in situ. The initial phase of the modification of proteins in the human lens by UV filters may be a dynamic process. In lenses, Cys residues of crystallins modify preferentially, but these adducts also decompose to release deaminated Kyn. This can then potentially react with other amino acids. Glutathione, which is present in high concentrations in the lenses of young people, may play a vital role in keeping proteins free from modification by intercepting reactive deaminated kynurenines formed by the spontaneous breakdown of free UV filters, promoting the decomposition of Kyn-Cys residues, and sequestering the unsaturated ketones once they are released from modified proteins.

Identification of the new UV filter compound cysteine-L-3-hydroxykynurenine O-beta-d-glucoside in human lenses.

UV filters protect the human lens and retina from UV light-induced damage. Here, we report the identification of a new UV filter, cysteine-l-3-hydroxykynurenine O-beta-d-glucoside, which is present in older normal human lenses. Its structure was confirmed by independent synthesis. It is likely this novel UV filter is formed in the lens by nucleophilic attack of cysteine on the unsaturated ketone derived from deamination of 3-hydroxykynurenine O-beta-d-glucoside. Quantitation studies revealed considerable variation in normal lens levels that may be traced to the marked instability of the cysteine adduct. The novel UV filter was not detected in advanced nuclear cataract lenses.