Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor.

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

Improving the kinetic parameters of nicotine oxidizing enzymes by homologous structure comparison and rational design.

Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high kcat or low Km, but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high kcat (>1 s-1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar Km for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by ∼25% (0.011 s-1 to 0.014 s-1), and reduce the Km of the NctB protein by approximately 34% (940 µM-622 µM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications.

An active site mutation in 6-hydroxy-l-Nicotine oxidase from Arthrobacter nicotinovorans changes the substrate specificity in favor of (S)-nicotine.

The enzyme 6-Hydroxy-l-Nicotine oxidase (HLNO) is a flavin-dependent enzyme that catalyzes the first step in the pyridine pathway of oxidation of nicotine as a source of energy and nitrogen in several bacteria. Recombinant Arthrobacter nicotinovorans HLNO also catalyzes oxidation of (s)-nicotine at a low but measurable rate (Fitzpatrick et al., 2016, Biochemistry 55, 697-703). Rational design and bioinformatics approaches, based on the known high-resolution structure of this enzyme (RCSB: 3NG7), were employed to further enhance the catalytic turnover and stability of the enzyme using (S)-nicotine as substrate. The active site residue Tyr311 forms a hydrogen bond with the hydroxyl group of (S)-6-OH-nicotine within the catalytic pocket. Its replacement by a tryptophan residue reduced the kcat for (S)-6-OH-nicotine by more than 6-fold and increased ~1.5-fold. Combining this mutation with two surface mutations that were predicted to enhance enzyme stability, further increased the kcat for nicotine resulting in a comparatively robust oxidation of (s)-nicotine (kcat >1 s-1) at 37 °C, at the same time reducing the specificity for (S)-OH-nicotine (kcat/KM) by more than 100-fold and increasing that for (S)-nicotine by more than 2-fold. Interestingly, adding a maltose-binding protein (MBP) tag onto the N-terminus of HLNO markedly increased the thermal stability of the enzyme, extending the half-life at 37 °C from ~2 h to ~22 h. This effect was due almost entirely to increased FAD retention, an observation that may prove useful to improve flavin retention in other flavin-dependent monoamine oxidases.

Cyclopropene-Templated Assembly of Medium Cycles via Ru-Catalyzed Ring-Closing Metathesis.

An expeditious click-click cyclize strategy for the assembly of medium-sized heterocyclic rings is described. The sequence involves the reaction of cycloprop-2-ene carboxylic acids with unsaturated amines to furnish amides, which are further subjected to a Cu-catalyzed directed carbomagnesiation and a ring-closing olefin metathesis reaction. This methodology allows for the efficient preparation of lactams with ring sizes up to 10.

Facile assembly of 1,5-diazocan-2-ones via cyclization of tethered sulfonamides to cyclopropenes.

The sulfonamide moiety was evaluated as an activating and stabilizing functional group in the metal-templated strain release-driven intramolecular nucleophilic addition of amines to cyclopropenes to generate 1,5-diazocan-2-ones.

Lactate as a biomarker for sleep.

STUDY OBJECTIVES: An ideal biomarker for sleep should change rapidly with sleep onset, remain at a detectably differential level throughout the sleep period, and exhibit a rapid change with waking. Currently, no molecular marker has been identified that exhibits all three properties. This study examined three substances (lactate, glucose, and glutamate) for suitability as a sleep biomarker. DESIGN: Using amperometric biosensor technology in conjunction with electroencephalograph (EEG) and electromyograph (EMG) monitoring, extracellular concentrations of lactate and glucose (Cohort 1) as well as lactate and glutamate (Cohort 2) were recorded over multiple sleep/wake cycles. PATIENTS OR PARTICIPANTS: There were 12 C57Bl/6J male mice (3-5 mo old). INTERVENTIONS: Sleep and waking transitions were identified using EEG recordings. Extracellular concentrations of lactate, glucose, and glutamate were evaluated before and during transition events as well as during extended sleep and during a 6-h sleep deprivation period. MEASUREMENTS AND RESULTS: Rapid and sustained increases in cortical lactate concentration (approximately 15 µM/min) were immediately observed upon waking and during rapid eye movement sleep. Elevated lactate concentration was also maintained throughout a 6-h period of continuous waking. A persistent and sustained decline in lactate concentration was measured during nonrapid eye movement sleep. Glutamate exhibited similar patterns, but with a much slower rise and decline (approximately 0.03 µM/min). Glucose concentration changes did not demonstrate a clear correlation with either sleep or wake. CONCLUSIONS: These findings indicate that extracellular lactate concentration is a reliable sleep/wake biomarker and can be used independently of the EEG signal.

Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036.

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.

The ABL switch control inhibitor DCC-2036 is active against the chronic myeloid leukemia mutant BCR-ABLT315I and exhibits a narrow resistance profile.

Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABL(T315I) mutant is highly resistant to imatinib, nilotinib, and dasatinib, and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor that binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABL(T315I) and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABL(T315I) enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC(50): 19 nmol/L), BCR-ABL(T315I) (IC(50): 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABL(T315I) to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABL(E255V / T315I) as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.

Simultaneous real-time measurement of EEG/EMG and L-glutamate in mice: A biosensor study of neuronal activity during sleep.

We report on electroencephalograph (EEG) and electromyograph (EMG) measurements concurrently with real-time changes in L-glutamate concentration. These data reveal a link between sleep state and extracellular neurotransmitter changes in a freely-moving (tethered) mouse. This study reveals, for the first time in mice, that the extracellular L-glutamate concentration in the pre-frontal cortex (PFC) increases during periods of extended wakefulness, decreases during extended sleep episodes and spikes during periods of REM sleep. Individual sleep epochs (10 s in duration) were scored as wake, slow-wave (SW) sleep or rapid eye movement (REM) sleep, and then correlated as a function of time with measured changes in L-glutamate concentrations. The observed L-glutamate levels show a statistically significant increase of 0.86 ± 0.26 µM (p < 0.05) over 37 wake episodes recorded from all mice (n = 6). Over the course of 49 measured sleep periods longer than 15 min, L-glutamate concentrations decline by a similar amount (0.88 ± 0.37 µM, p < 0.08). The analysis of 163 individual REM sleep episodes greater than one min in length across all mice (n = 6) demonstrates a significant rise in L-glutamate levels as compared to the 1 min preceding REM sleep onset (RM-ANOVA, DF = 20, F = 6.458, p < 0.001). The observed rapid changes in L-glutamate concentration during REM sleep last only between 1 and 3 min. The approach described can also be extended to other regions of the brain which are hypothesized to play a role in sleep. This study highlights the importance of obtaining simultaneous measurements of neurotransmitter levels in conjunction with sleep markers to help elucidate the underlying physiological and ultimately the genetic components of sleep.

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

Gram-scale syntheses of the (1-->3)-linked and (1-->4)-linked hyaluronan disaccharides.

The first gram-scale syntheses of two hyaluronan disaccharides are described. Construction of the (1-->4)-linked disaccharide 12 was achieved in 12% overall yield using 2,3-bis-dimethyl acetal protection in combination with chlorosilane-induced carbamate cleavage methodologies. The uronic acid functionality was installed using TEMPO oxidation with NaOCl as the hypochlorite source. The (1-->3)-linked disaccharide 18 was achieved in 7% overall yield utilizing acetonide protection in addition to the chlorosilane-induced carbamate cleavage methodology and the TEMPO oxidation.

Synthesis of Lipophilic Paramagnetic Contrast Agents.

The facile, high-yielding synthesis of a series of macrocycles 7a-k in 75-100% yield is reported. The transformation of these compounds to their carboxymethylated analogues 8a-k in 75-90% yield and subsequent gadolinium complexes 9a-k provides a series of homologous neutral paramagnetic contrast agents (PCAs) with tunable lipophilicity. Alkylated cationic intermediates 6a-k are prepared in yields of 72-94% from glyoxal adduct of cyclen (5) and slight excesses of alkyl iodides. The methodology is selective for monoalkylation and amenable to large-scale synthesis.