Biomedical subjects

Richard B Silverman

Publications and source records attributed to Richard B Silverman.

At least 19 recordsLinked to original sources

PubMed2026

Proteomic analysis reveals early pathological defects in corticospinal motor neurons of a spastin model of hereditary spastic paraplegia, which are improved by NU-9 treatment.

Upper motor neuron (UMN) degeneration is a characteristic feature of hereditary spastic paraplegia (HSP), a genetically heterogeneous heritable neurodegenerative disorder resulting from mutations in over ninety genes. The mutations in the SPAST gene, which encodes the microtubule-severing protein spastin, are responsible for about 40% of all HSP cases. To date, the cellular and molecular mechanisms linking mutant spastin protein to UMN vulnerability in HSP patients remain unknown and there are no disease modifying therapies. To address this knowledge gap, we isolated pure populations of corticospinal motor neurons (CSMN; a.k.a. UMN in mice) from SPASTC448Y-UeGFP reporter mice at two pre-symptomatic time points and performed bottom-up proteomic analyses to reveal changes in their proteome that informs the underlying causes of their initial vulnerability. We find dynamic changes in their proteome and that limitations with cytoarchitectural integrity and stability of key organelles contribute to their neuronal vulnerability. Since the compound NU-9 was shown to improve similar cellular problems in CSMN that are diseased due to misfolded SOD1 toxicity and TDP-43 pathology, we further investigated its effect on the well-established pathological features of HSP that are recapitulated in the SPASTC448Y mice. We find that NU-9 treatment (100 mg/kg, for 100 days) significantly prevented degeneration of corticospinal axons, restored the integrity of mitochondria and endoplasmic reticulum, and reduced the presence of electron-dense accumulations in the CSMN of SPASTC448Y mice.

Animals↗

PubMed2007

Electronic effects of peripheral substituents at porphyrin meso positions.

Porphyrins are stable molecules with a macrocyclic conjugated system and often peripheral substituents. This unique structure makes the electronic properties of the four meso-carbons (the methine bridges) nearly identical. Replacement of the weakly electron-polarizing 2,4-vinyl groups of protoporphyrin IX with strongly electron-polarizing acetyl groups not only leads to much lower meso-carbon reactivities toward electrophilic aromatic substitution but also results in a significant meso-selectivity (the beta- and gamma-meso-positions become much more nucleophilic (basic) than the alpha- and delta-meso-positions). To further investigate the relationship between the porphyrin meso-carbon reactivities and the peripheral substituents, two monoacetylporphyrin analogues also were synthesized. This investigation not only leads to empirical rules for predicting porphyrin meso-carbon selectivities but also provides important models for theoretical calculations of porphyrin aromaticity.

Electrons↗

PubMed2006

Fluorinated conformationally restricted gamma-aminobutyric acid aminotransferase inhibitors.

On the basis of the structures of several potent inhibitor molecules for gamma-aminobutryric acid aminotransferase (GABA-AT) that were previously reported, six modified fluorine-containing conformationally restricted analogues were designed, synthesized, and tested as GABA-AT inhibitors. The syntheses of all six molecules followed from a readily synthesized ketone intermediate. Three of the molecules were found to be irreversible inhibitors of GABA-AT with comparable or larger k(inact)/K(I) values than that of vigabatrin, a clinically used antiepilepsy drug, and the other three were reversible inhibitors. A possible mechanism for inactivation by one of the inactivators is proposed.

4-Aminobutyrate Transaminase↗

PubMed2006

(+/-)-(1S,2R,5S)-5-Amino-2-fluorocyclohex-3-enecarboxylic acid. A potent GABA aminotransferase inactivator that irreversibly inhibits via an elimination-aromatization pathway.

Inhibition of gamma-aminobutyric acid aminotransferase (GABA-AT) increases the concentration of GABA, an inhibitory neurotransmitter in human brain, which could have therapeutic applications for a variety of neurological diseases, including epilepsy. On the basis of studies of several previously synthesized conformationally restricted GABA-AT inhibitors, (+/-)-(1S,2R,5S)-5-amino-2-fluorocyclohex-3-enecarboxylic acid (12) was designed as a mechanism-based inactivator. This compound was shown to irreversibly inhibit GABA-AT; substrate protects the enzyme from inactivation. Mechanistic experiments demonstrated the loss of one fluoride ion per active site during inactivation and the formation of N-m-carboxyphenylpyridoxamine 5'-phosphate (26), the same product generated by inactivation of GABA-AT by gabaculine (8). An elimination-aromatization mechanism is proposed to account for these results.

4-Aminobutyrate Transaminase↗

PubMed2006

Conformationally restricted dipeptide amides as potent and selective neuronal nitric oxide synthase inhibitors.

Four new conformationally restricted analogues of a potent and selective neuronal nitric oxide synthase inhibitor, l-nitroargininyl-l-2,4-diaminobutyramide (1), have been synthesized. N(alpha)-Methyl and N(alpha)-benzyl derivatives (3 and 4, respectively) of 4-N-(l-Arg(NO(2))-trans-4-amino-l-prolineamide (2) are also selective inhibitors, but the potency and selectivity of 3 are weak. Analogue 4 has only one-third the potency and one-half to one-third the selectivity of 2 against iNOS (inducible nitric oxide synthase) and eNOS (endothelial nitric oxide synthase), respectively. 3-N-(l-Arg(NO)(2))-trans-3-amino-l-prolineamide (6) is as potent an inhibitor of nNOS (neuronal nitric oxide synthase) as 2; selectivity for nNOS over iNOS is half of that for 2, but the selectivity for nNOS over eNOS is almost double that for 2. The corresponding cis-isomer (5) is a weak inhibitor of nNOS. These results are supported by computer modeling.

Amides↗

PubMed2006

Hydroxyl-terminated peptidomimetic inhibitors of neuronal nitric oxide synthase.

The X-ray structure of previously studied dipeptidomimetic inhibitors bound in the active site of neuronal nitric oxide synthase (nNOS) presented a possibility for optimizing the strength of enzyme-inhibitor interactions as well as for enhancing bioavailability. These desirable properties may be attainable by replacement of the terminal amino group of the parent compounds (1-6) with a hydroxyl group (11-13, and 18-20). The hypothesized effect would be twofold: first, a change from a positively charged amino group to a neutral hydroxyl group might afford more drug-like character and blood-brain barrier permeability to the inhibitors; second, as suggested by docking studies, the incorporated hydroxyl group might displace an active site water molecule with which the terminal amino group of the original compounds indirectly hydrogen bonds. In vitro activity assays of the hydroxyl-terminated analogs (11-13 and 18-20) showed greater than an order of magnitude increase in K(i) values (decreased potency) relative to the amino-terminated compounds. These experimental data support the importance to enzyme binding of a potential electrostatic interaction relative to a hydrogen bonding interaction.

Animals↗

PubMed2006

Carbonyl- and sulfur-containing analogs of suberoylanilide hydroxamic acid: Potent inhibition of histone deacetylases.

Suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase, is used in clinical trials for a variety of advanced cancers. Twelve new analogs of SAHA were synthesized and tested as in vitro inhibitors of isolated histone deacetylases (HDACS) and in vivo inhibitors of interferon regulated transcriptional responses (a marker for HDAC activity). The analogs containing an alpha-mercaptoketone or an alpha-thioacetoxyketone were more potent than SAHA in both assays.

Cells, Cultured↗

PubMed2006

Design, synthesis, and biological testing of potential heme-coordinating nitric oxide synthase inhibitors.

Based on computer modeling of the active site of nitric oxide synthases (NOS), a series of 10 amidine compounds (9-18) was designed including potential inhibitors that involve the coordination of side-chain functional groups with the iron of the heme cofactor. The most potent and selective compound was the methylthio amidine analogue 9, which was more potent than L-nitroarginine with 185-fold selectivity for inhibition of neuronal NOS over endothelial NOS. It also exhibited time-dependent inhibition, but did not involve the mechanism previously proposed for other amidine inhibitors of NOS. None of the compounds, however, exhibited heme-binding characteristics according to absorption spectroscopy.

Animals↗

PubMed2006

A novel peptide sansalvamide analogue inhibits pancreatic cancer cell growth through G0/G1 cell-cycle arrest.

Patients with pancreatic cancer have little hope for cure because no effective therapies are available. Sansalvamide A is a cyclic depsipeptide produced by a marine fungus. We investigated the effect of a novel sansalvamide A analogue on growth, cell-cycle phases, and induction of apoptosis in human pancreatic cancer cells in vitro. The sansalvamide analogue caused marked time- and concentration-dependent inhibition of DNA synthesis and cell proliferation of two human pancreatic cancer cell lines (AsPC-1 and S2-013). The analogue induced G0/G1 phase cell-cycle arrest and morphological changes suggesting induction of apoptosis. Apoptosis was confirmed by annexin V binding. This novel sansalvamide analogue inhibits growth of pancreatic cancer cells through G0/G1 arrest and induces apoptosis. Sansalvamide analogues may be valuable for the treatment of pancreatic cancer.

Antineoplastic Agents↗

PubMed2005

Syntheses and evaluation of fluorinated conformationally restricted analogues of GABA as potential inhibitors of GABA aminotransferase.

Inhibition of gamma-aminobutyric acid aminotransferase (GABA-AT) could raise the concentration of GABA, an inhibitory neurotransmitter in the human brain, and could have therapeutic applications for a variety of neurological diseases including epilepsy. Four fluorine-containing analogues of GABA with conformations restricted by a cyclohexane ring system were designed and synthesized, but unlike some of their five-membered ring counterparts, minimal inhibition of GABA-AT was observed. It is likely that the rigid chair conformation of these compounds cannot be accommodated well in the enzyme's active site.

4-Aminobutyrate Transaminase↗

PubMed2005

Exploring the binding conformations of bulkier dipeptide amide inhibitors in constitutive nitric oxide synthases.

A series of L-nitroarginine-based dipeptide inhibitors are highly selective for neuronal nitric oxide synthase (nNOS) over the endothelial isoform (eNOS). Crystal structures of these dipeptides bound to both isoforms revealed two different conformations, curled in nNOS and extended in eNOS, corresponding to higher and lower binding affinity to the two isoforms, respectively. In previous studies we found that the primary reason for selectivity is that Asp597 in nNOS, which is Asn368 in eNOS, provides greater electrostatic stabilization in the inhibitor complex. While this is the case for smaller dipeptide inhibitors, electrostatic stabilization may no longer be the sole determinant for isoform selectivity with bulkier dipeptide inhibitors. Another residue farther away from the active site, Met336 in nNOS (Val106 in eNOS), is in contact with bulkier dipeptide inhibitors. Double mutants were made to exchange the D597/M336 pair in nNOS with N368/V106 in eNOS. Here we report crystal structures and inhibition constants for bulkier dipeptide inhibitors bound to nNOS and eNOS that illustrate the important role played by residues near the entry to the active site in isoform selective inhibition.

Animals↗

PubMed2005

New substrates and inhibitors of gamma-aminobutyric acid aminotransferase containing bioisosteres of the carboxylic acid group: design, synthesis, and biological activity.

A series of potential substrates of gamma-aminobutyric acid aminotransferase (GABA-AT) with lipophilic bioisosteres of the carboxylic acid group (2-7) were synthesized and tested. Most of the synthesized compounds showed substrate activities with GABA-AT; 1H-tetrazole-5-propanamine (6) was the best of those tested. The potential time-dependent inhibitor of GABA-AT, 1H-tetrazole-5-(alpha-vinyl-propanamine) (8), was designed based on the structures of 6 and the antiepilepsy drug vigabatrin (4-aminohex-5-enoic acid, 1). The synthesized compound 8 showed time-dependent inhibition of GABA-AT, but its potency is lower than that of vigabatrin. Methylation of the tetrazole group in 8 resulted in loss of time-dependent activity, suggesting that the tetrazole ring, the carboxylate bioisostere, exists in its deprotonated form in the enzyme active site.

4-Aminobutyrate Transaminase↗

PubMed2005

Enantiomers of cis-constrained and flexible 2-substituted GABA analogues exert opposite effects at recombinant GABA(C) receptors.

The effects of the enantiomers of a number of flexible and cis-constrained GABA analogues were tested on GABA(C) receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. (1S,2R)-cis-2-Aminomethylcyclopropane-1-carboxylic acid ((+)-CAMP), a potent and full agonist at the rho1 (EC(50) approximately 40 microM, I(max) approximately 100%) and rho 2 (EC(50) approximately 17 microM, I(max) approximately 100%) receptor subtypes, was found to be a potent partial agonist at rho3 (EC(50) approximately 28 microM, I(max) approximately 70%). (1R,2S)-cis-2-Aminomethylcyclopropane-1-carboxylic acid ((-)-CAMP), a weak antagonist at human rho1 (IC(50) approximately 890 microM) and rho2 (IC(50) approximately 400 microM) receptor subtypes, was also found to be a moderately potent antagonist at rat rho3 (IC(50) approximately 180 microM). Similarly, (1R,4S)-4-aminocyclopent-2-ene-1-carboxylic acid ((+)-ACPECA) was a full agonist at rho1 (EC(50) approximately 135 microM, I(max) approximately 100%) and rho2 (EC(50) approximately 60 microM, I(max) approximately 100%), but only a partial agonist at rho3 (EC(50) approximately 112 microM, I(max) approximately 37%), while (1S,4R)-4-aminocyclopent-2-ene-1-carboxylic acid ((-)-ACPECA) was a weak antagonist at all three receptor subtypes (IC(50)>>300 microM). 4-Amino-(S)-2-methylbutanoic acid ((S)-2MeGABA) and 4-amino-(R)-2-methylbutanoic acid ((R)-2MeGABA) followed the same trend, with (S)-2MeGABA acting as a full agonist at the rho1 (EC(50) approximately 65 microM, I(max) approximately 100%), and rho2 (EC(50) approximately 20 microM, I(max) approximately 100%) receptor subtypes, and a partial agonist at rho3 (EC(50) approximately 25 microM, I(max) approximately 90%). (R)-2MeGABA, however, was a moderately potent antagonist at all three receptor subtypes (IC(50) approximately 16 microM at rho1, 125 microM at rho2 and 35 microM at rho3). On the basis of these expanded biological activity data and the solution-phase molecular structures obtained at the MP2/6-31+G* level of ab initio theory, a rationale is proposed for the genesis of this stereoselectivity effect.

Animals↗

PubMed2005

N-methylsansalvamide a peptide analogues. Potent new antitumor agents.

Sansalvamide A, a cyclic depsipeptide isolated from a marine fungus of the genus Fusarium, is composed of four hydrophobic amino acids (Phe, two Leu, Val) and one hydroxy acid ((S)-2-hydroxy-4-methylpentanoic acid; O-Leu) with five stereogenic centers all having S-stereochemistry. We have recently synthesized the corresponding cyclic peptide (Gu, W.; Liu, S.; Silverman, R. B. Organic Lett. 2002, 4, 4171-4174) and found that it too has antitumor activity. N-Methylation can enhance potency and selectivity for peptides. Consequently, here we synthesize 12 different N-methylated sansalvamide A peptide analogues and show that for several different tumor cell lines three of these analogues are more potent than the natural product; in pancreatic cells, sansalvamide A shows little activity, but the N-methylsansalvamide peptides are potent cytotoxic agents.

Antineoplastic Agents↗

PubMed2005

Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines.

Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol.2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased k(cat)/K(m) values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-alpha-methylbenzylamine (N-CalphaMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CalphaMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2005

Mechanism of inactivation of inducible nitric oxide synthase by amidines. Irreversible enzyme inactivation without inactivator modification.

Nitric oxide synthases (NOS) are hemoproteins that catalyze the reaction of L-arginine to L-citrulline and nitric oxide. N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was reported to be a slow, tight-binding, and highly selective inhibitor of iNOS in vitro and in vivo. Previous mechanistic studies reported that 1400W was recovered quantitatively after iNOS fully lost its activity and modification to iNOS was not detected. Here, it is shown that 1400W is a time-, concentration-, and NADPH-dependent irreversible inactivator of iNOS. HPLC-electrospray mass spectrometric analysis of the incubation mixture of iNOS with 1400W shows both loss of heme cofactor and formation of biliverdin, as was previously observed for iNOS inactivation by another amidine-containing compound, N5-(1-iminoethyl)-L-ornithine (L-NIO). The amount of biliverdin produced corresponds to the amount of heme lost by 1400W inactivation of iNOS. A convenient MS/MS-HPLC methodology was developed to identify the trace amount of biliverdin produced by inactivation of iNOS with either 1400W or L-NIO to be biliverdin IXalpha out of the four possible regioisomers. Two mechanisms were previously proposed for iNOS inactivation by L-NIO: (1) uncoupling of the heme peroxide intermediate, leading to destruction of the heme to biliverdin; (2) abstraction of a hydrogen atom from the amidine methyl group followed by attachment to the heme cofactor, which causes the enzyme to catalyze the heme oxygenase reaction. The second mechanistic proposal was ruled out by inactivation of iNOS with d3-1400W, which produced no d2-1400W. Detection of carbon monoxide as one of the heme-degradation products further excludes the covalent heme adduct mechanism. On the basis of these results, a third mechanism is proposed in which the amidine inactivators of iNOS bind as does substrate L-arginine, but because of the amidine methyl group, the heme peroxy intermediate cannot be protonated, thereby preventing its conversion to the heme oxo intermediate. This leads to a change in the enzyme mechanism to one that resembles that of heme oxygenase, an enzyme known to convert heme to biliverdin IXalpha. This appears to be the first example of a compound that causes irreversible inactivation of an enzyme without itself becoming modified in any way.

Amidines↗

PubMed2005

Selective neuronal nitric oxide synthase inhibitors.

This review includes the non-patent literature up to October 2004 that deals with selective neuronal nitric oxide synthase inhibitors (highest potency is for the neuronal isozyme). Some non-selective inhibitors or selective inducible nitric oxide synthase inhibitors are mentioned if they are related to compounds that are discussed; structures of these compounds generally are not given. In vitro inhibition constants are given either as IC(50) values or as K(i)values. An IC(50) value, the inhibitor concentration that produces 50% inhibition in the presence of a constant concentration of substrate, is obtained by extrapolation of several rate data points to 50% inhibition. K(i) values are derived from several types of plots that relate the concentration of inhibitor with enzyme velocity in the presence of a variety of substrate concentrations [1]. The K(i) value can be estimated from the IC(50) value [2]. Although the two inhibition constants are related, they are not the same; generally, the reported K(i) values tend to be lower than the IC(50) values. If specifics are desired about how the data were collected, then the reader will have to look in the literature cited. No attempt was made to be exhaustive in citing all references related to specific inhibitors; rather, examples of literature references are given for each inhibitor described.

Animals↗

PubMed2004

Mechanistic crystallography. Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid as elucidated by crystallography.

(1R,3S,4S)-3-Amino-4-fluorocyclopentane-1-carboxylic acid (7) was previously shown to be a mechanism-based inactivator of gamma-aminobutyric acid aminotransferase (GABA-AT) [Qiu, J. and Silverman, R. B. (2000) J. Med. Chem. 43, 706-720]. Two mechanisms were considered as reasonable possibilities, a Michael addition mechanism and an enamine mechanism. On the basis of a variety of chemical studies, including tedious radiolabeling experiments, it was concluded that inactivation by 7 proceeds by a Michael addition mechanism. Here, a crystal structure of 7 bound to pig liver GABA-AT is reported, which clearly demonstrates that the adduct formed is derived from an enamine mechanism. This represents another example of how crystallography is an important tool for elucidation of inactivation mechanisms.

4-Aminobutyrate Transaminase↗