Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for \u3ci\u3eβ\u3c/i\u3e-elimination function

There is currently great interest in human serine racemase, the enzyme responsible for producing the NMDA co-agonist D-serine. Reported correlation of D-serine levels with disorders including Alzheimer’s disease, ALS, and ischemic brain damage (elevated D-serine) and schizophrenia (reduced D-serine) has further piqued this interest. Reported here is a structure/activity relationship study of position Ser84, the putative re-face base. In the most extreme case of functional reprogramming, the S84D mutant displays a dramatic reversal of β-elimination substrate specificity in favor of L-serine over the normally preferred L-serine-O-sulfate (~1200-fold change in kcat/Km ratios) and L (L-THA; ~5000-fold change in kcat/Km ratios) alternative substrates. On the other hand, the S84T (which performs L-Ser racemization activity), S84A (good kcat but high Km for L-THA elimination), and S84N mutants (nearly WT efficiency for L-Ser elimination) displayed intermediate activity, all showing a preference for the anionic substrates, but generally attenuated compared with the native enzyme. Inhibition studies with L-erythro-β-hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with Ki = 31 ± 1.5 μM and 1.5 ± 0.1mM, respectively, and the S84D being inert to inhibition. Computational modeling pointed to a key role for residue Arg-135 in binding and properly positioning the L-THA and L-serine-O-sulfate substrates and the L-erythro-β-hydroxyaspartate inhibitor. Examination of available sequence data suggests that Arg-135 may have originated for L-THA-like-β-elimination function in earlier evolutionary variants, and examination of available structural data suggests that a Ser84-H2O-Lys114 hydrogen-bonding network in human serine racemase lowers the pKa of the Ser84 re-face base

“Zipped Synthesis” by Cross-Metathesis Provides a Cystathionine β‑Synthase Inhibitor that Attenuates Cellular H\u3csub\u3e2\u3c/sub\u3eS Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model

The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a “zipped” approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C2-symmetric CBS product (L,L)-cystathionine. The “zipped” concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine−imine interchange. It is demonstrated that the most potent “zipped” inhibitor 6S reduces H2S production in SHSY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia. Supplementary information (112 pp.) is attached (below)

“Zipped Synthesis” by Cross-Metathesis Provides a Cystathionine β‑Synthase Inhibitor that Attenuates Cellular H\u3csub\u3e2\u3c/sub\u3eS Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model

The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a “zipped” approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C2-symmetric CBS product (L,L)-cystathionine. The “zipped” concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine−imine interchange. It is demonstrated that the most potent “zipped” inhibitor 6S reduces H2S production in SHSY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia. Supplementary information (112 pp.) is attached (below)

An Integrated Study of PLP-Dependent Enzyme Mechanisms Through Targeted Mutagenesis, Inhibitor Design and Kinetic Evaluation

The research in Chapters 1 and 2 focuses on developing inactivators of pyridoxal-5’-phosphate (PLP) enzymes. The work within Chapter 1 contributes to a methodology that facilitates the introduction of a methoxyvinyl group into a variety of systems with the future potential of developing PLP focused inactivators inspired by the natural product methoxyvinylglycine. The work within Chapter 2 describes the first synthesis of b,b-difluorovinyl phenyl sulfone, a highly sought after, but elusive, electrophile that we demonstrate also serves as a (1’-fluoro)vinyl cation equivalent. This electrophile is utilized to synthesize quaternary α-(1’-fluoro)vinyl amino acids, bearing the native side chains of eight amino acids. The α-(1’-fluoro)vinyl analogue of lysine is shown to irreversibly inactivate the model enzyme lysine decarboxylase. This new inhibitor class has the potential to target a large variety of PLP-dependent enzymes. Chapter 3 turns to studies directed at developing inhibitors for an important PLP enzyme, cystathionine β-synthase (CBS). CBS is a β-eliminase/replacement enzyme that catalyzes L,L-cystathionine biosynthesis, a key step in the transulfuration pathway. CBS is also responsible for the production of H2S as a “gaseous hormone” in the brain. CBS overexpression pursuant to ischemic stroke appears to be a key element leading to neuronal cell damage. The focus here was to synthesize C2-symmetric cystathionine-based mimics to effectively inhibit the enzyme and test for increased cell viability in stroke model systems. A fully saturated hydrazino-mimic showed the strongest inhibition of the compounds tested in vitro. This newly found inhibitor led to ~ 70% reduction of infarct volume upon ICV administration 1 h post-occlusion in a rat model for stroke. Chapter 4 focuses on another enzyme associated with neuronal signaling, serine racemase, which produces D-serine, a co-agonist of the NMDA receptor associated with learning and memory. Site-directed mutagenesis of the active site re-face base (S84D, S84N, S84T) coupled with steady enzyme kinetics on a set of substrates and inhibitors revealed dramatic changes in substrate specificity seen as a function of re-face base. Molecular modeling and docking allowed one to rationalize these effects and suggests key roles for substrate positioning and stereoelectronics in this active site

An Integrated Study of PLP-Dependent Enzyme Mechanisms Through Targeted Mutagenesis, Inhibitor Design and Kinetic Evaluation

The research in Chapters 1 and 2 focuses on developing inactivators of pyridoxal-5’-phosphate (PLP) enzymes. The work within Chapter 1 contributes to a methodology that facilitates the introduction of a methoxyvinyl group into a variety of systems with the future potential of developing PLP focused inactivators inspired by the natural product methoxyvinylglycine. The work within Chapter 2 describes the first synthesis of b,b-difluorovinyl phenyl sulfone, a highly sought after, but elusive, electrophile that we demonstrate also serves as a (1’-fluoro)vinyl cation equivalent. This electrophile is utilized to synthesize quaternary α-(1’-fluoro)vinyl amino acids, bearing the native side chains of eight amino acids. The α-(1’-fluoro)vinyl analogue of lysine is shown to irreversibly inactivate the model enzyme lysine decarboxylase. This new inhibitor class has the potential to target a large variety of PLP-dependent enzymes. Chapter 3 turns to studies directed at developing inhibitors for an important PLP enzyme, cystathionine β-synthase (CBS). CBS is a β-eliminase/replacement enzyme that catalyzes L,L-cystathionine biosynthesis, a key step in the transulfuration pathway. CBS is also responsible for the production of H2S as a “gaseous hormone” in the brain. CBS overexpression pursuant to ischemic stroke appears to be a key element leading to neuronal cell damage. The focus here was to synthesize C2-symmetric cystathionine-based mimics to effectively inhibit the enzyme and test for increased cell viability in stroke model systems. A fully saturated hydrazino-mimic showed the strongest inhibition of the compounds tested in vitro. This newly found inhibitor led to ~ 70% reduction of infarct volume upon ICV administration 1 h post-occlusion in a rat model for stroke. Chapter 4 focuses on another enzyme associated with neuronal signaling, serine racemase, which produces D-serine, a co-agonist of the NMDA receptor associated with learning and memory. Site-directed mutagenesis of the active site re-face base (S84D, S84N, S84T) coupled with steady enzyme kinetics on a set of substrates and inhibitors revealed dramatic changes in substrate specificity seen as a function of re-face base. Molecular modeling and docking allowed one to rationalize these effects and suggests key roles for substrate positioning and stereoelectronics in this active site

Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for \u3ci\u3eβ\u3c/i\u3e-elimination function

There is currently great interest in human serine racemase, the enzyme responsible for producing the NMDA co-agonist D-serine. Reported correlation of D-serine levels with disorders including Alzheimer’s disease, ALS, and ischemic brain damage (elevated D-serine) and schizophrenia (reduced D-serine) has further piqued this interest. Reported here is a structure/activity relationship study of position Ser84, the putative re-face base. In the most extreme case of functional reprogramming, the S84D mutant displays a dramatic reversal of β-elimination substrate specificity in favor of L-serine over the normally preferred L-serine-O-sulfate (~1200-fold change in kcat/Km ratios) and L (L-THA; ~5000-fold change in kcat/Km ratios) alternative substrates. On the other hand, the S84T (which performs L-Ser racemization activity), S84A (good kcat but high Km for L-THA elimination), and S84N mutants (nearly WT efficiency for L-Ser elimination) displayed intermediate activity, all showing a preference for the anionic substrates, but generally attenuated compared with the native enzyme. Inhibition studies with L-erythro-β-hydroxyaspartate follow this trend, with both WT serine racemase and the S84N mutant being competitively inhibited, with Ki = 31 ± 1.5 μM and 1.5 ± 0.1mM, respectively, and the S84D being inert to inhibition. Computational modeling pointed to a key role for residue Arg-135 in binding and properly positioning the L-THA and L-serine-O-sulfate substrates and the L-erythro-β-hydroxyaspartate inhibitor. Examination of available sequence data suggests that Arg-135 may have originated for L-THA-like-β-elimination function in earlier evolutionary variants, and examination of available structural data suggests that a Ser84-H2O-Lys114 hydrogen-bonding network in human serine racemase lowers the pKa of the Ser84 re-face base

“Zipped Synthesis” by Cross-Metathesis Provides a Cystathionine β‑Synthase Inhibitor that Attenuates Cellular H2S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model

[Image: see text] The gaseous neuromodulator H(2)S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H(2)S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a “zipped” approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C(2)-symmetric CBS product (l,l)-cystathionine. The “zipped” concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine–imine interchange. It is demonstrated that the most potent “zipped” inhibitor 6S reduces H(2)S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia

“Zipped Synthesis” by Cross-Metathesis Provides a Cystathionine β‑Synthase Inhibitor that Attenuates Cellular H₂S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model

The gaseous neuromodulator H₂S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H₂S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a “zipped” approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-<i>C</i>₂-symmetric CBS product (l,l)-cystathionine. The “zipped” concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine–imine interchange. It is demonstrated that the most potent “zipped” inhibitor <b>6S</b> reduces H₂S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor <b>6S</b> dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia