Biomedical subjects

Harold Kohn

Publications and source records attributed to Harold Kohn.

At least 19 recordsLinked to original sources

PubMed2005

The novel antiepileptic drug lacosamide blocks behavioral and brain metabolic manifestations of seizure activity in the 6 Hz psychomotor seizure model.

Brain metabolic activation after 6 Hz electrical stimulation (32 mA, 3s stimulus duration) was assessed by autoradiographic analysis of 14C-2-deoxyglucose (2-DG) uptake. In addition, effects of the new antiepileptic drug lacosamide were examined on the stimulation-induced metabolic activation. The 6 Hz stimulation via corneal electrodes induced a robust increase 2-DG uptake in cerebral cortical regions, lateral amygdala, and the caudate-putamen. Many other brain regions were not affected by the stimulation, including the hippocampal formation, medial nuclei of the amygdala, thalamus, and hypothalamus. Lacosamide (20 mg/kg) injected i.p. 30 min before application of electrical stimulation antagonized completely the seizure-induced brain metabolic activation but did not affect basal 2-DG uptake. The data provide evidence that lacosamide antagonizes the neural activation induced by an electrical seizure stimulus, without suppressing normal brain metabolic activity.

Acetamides↗

PubMed2005

Fluorine-substituted dihydrobicyclomycins: synthesis and biochemical and biological properties.

Many studies show that selective introduction of fluorine within pharmacological agents leads to improved activities. In this study, we determine the effects of aryl fluorine substitution in 5a-(benzylsulfanyl)-dihydrobicyclomycin (3) on the in vitro inhibition of Escherichia coli rho-dependent ATPase activity. Compound 3 is an analog of bicyclomycin (1), which is the only known selective inhibitor of rho, and 1 and 3 have comparable in vitro inhibitory activities. We demonstrate that aryl fluorine substitution of 3 leads to increase in inhibitory activity but that the beneficial effects due to fluorine were dependent upon the site and number of fluorine substituents. The bioactivities are rationalized in terms of the bond moment created by the aryl fluoride bond within the 5a-aryl dihydrobicyclomycin-rho-binding pocket. Use of this hypothesis led to the design of dihydrobicyclomycin derivatives with superior in vitro rho inhibitory activities.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed2005

Development of a technique to determine bicyclomycin-rho binding and stoichiometry by isothermal titration calorimetry and mass spectrometry.

Bicyclomycin (1) is the only natural product inhibitor of the transcription termination factor rho. Rho is a hexameric helicase that terminates nascent RNA transcripts utilizing ATP hydrolysis and is an essential protein for many bacteria. The paucity of information concerning the 1-rho interaction stems from the weak binding affinity of 1. We report a novel technique using imine formation with rho to enhance the affinity of a bicyclomycin analogue and determine the binding stoichiometry by isothermal titration calorimetry (ITC) and mass spectrometry (MS). Our designed bicyclomycin ligand, 5a-(3-formyl-phenylsulfanyl)-dihydrobicyclomycin (2) (apparent I(50) = 4 muM), inhibits rho an order of magnitude more efficiently than 1 (I(50) = 60 muM). MS shows that 2 selectively forms an imine with K181 in rho. We found that despite the heterogeneity of ATP binding (three tight and three weak) imposed on the rho hexamer, the nearby bicyclomycin binding pocket is not affected, and both 1 and 2 bind with equal affinity to all six subunits.

Adenosine Triphosphatases↗

PubMed2005

The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site.

We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.

Alanine Racemase↗

PubMed2005

Bismuth-dithiol inhibition of the Escherichia coli rho transcription termination factor.

Bismuth-dithiol mixtures are proven antimicrobial agents with unknown mechanism(s) of action. We show that select bismuth-dithiol solutions inhibit the Escherichia coli rho transcription termination factor. Rho is an essential enzyme in most Gram-negative prokaryotes and without rho function the cells are not viable. Bismuth complexes with 2,3-dimercapto-1-propanol (BiBAL) (3:1 solutions) functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay (I50=60 microM) and as a competitive inhibitor with respect to ribo(C)10 in the poly(dC)-ribo(C)10-dependent ATPase assay. The minimum inhibitory concentration (MIC) of bacterial growth for BiBAL (3:1) in the liquid culture assay using E. coli W3350 was 16 microM. Using the tnaA/lacZ fusion reporter assay we showed that sublethal amounts (3 microM) of BiBAL (3:1 solution) led to a small increase (37%) in in vivo beta-galactosidase activity in E. coli SVS1144, which corresponds to antitermination of the tna operon as a result of rho inhibition. We concluded that BiBAL was a potent in vitro rho inhibitor but its effect on in vivo rho processes was modest indicating that other mechanisms contributed to the antibacterial activity of BiBAL. Our study suggests that structural changes in the dithiol unit that provide greater bismuth binding may improve rho specificity, a macromolecular target not previously recognized for bismuth therapy.

Adenosine Triphosphatases↗

PubMed2005

7-N,7'-N'-(1",2"-Dithianyl-3",6"-dimethylenyl)bismitomycin C: synthesis and nucleophilic activation of a dimeric mitomycin.

Dimeric alkylating agents that modify complementary DNA strands have engendered significant interest. We have prepared the novel dimeric mitomycin, 7-N,7'-N'-(1",2"-dithianyl-3",6"-dimethylenyl)bismitomycin C (9), in which the mitomycins are bridged by a dithiane unit. Dimer 9, like the clinically tested acyclic disulfides KW-2149 (3) and BMS-181174 (4), was designed to activate under nucleophilic and reductive conditions. Successive nucleophile-mediated disulfide cleavage transformations of 9 are expected to generate thiol species ideally positioned to render the two mitomycin systems vulnerable to nucleophilic attack and permit DNA interstrand cross-link formation. The dithiane linker, strategically positioned between the two mitomycins, distinguished 9 from 3 and 4. Nucleophilic activation of this cyclic disulfide permitted both activated mitomycins to remain tethered to one another. We report the synthesis of 9, and show that the nucleophile Et(3)P markedly enhances the activation and consumption of 9, compared with the reference compound 7-N, 7"-N'-(cyclohexanyl-trans-1",4"-dimethylenyl)bismitomycin C (27). We further demonstrated that provides higher levels of DNA interstrand cross-links than either the dimeric reference compounds, and 7-N,7-N'-(2",5"-dihydroxy-1",6"-hexanediyl)bismitomycin C (28), or the monomeric mitomycins, 1 and 3, when Et(3)P is added to solutions containing EcoRI-linearized pBR322 DNA.

Binding Sites↗

PubMed2005

Structural mechanism of inhibition of the Rho transcription termination factor by the antibiotic bicyclomycin.

Rho is a hexameric RNA/DNA helicase/translocase that terminates transcription of select genes in bacteria. The naturally occurring antibiotic, bicyclomycin (BCM), acts as a noncompetitive inhibitor of ATP turnover to disrupt this process. We have determined three independent X-ray crystal structures of Rho complexed with BCM and two semisynthetic derivatives, 5a-(3-formylphenylsulfanyl)-dihydrobicyclomycin (FPDB) and 5a-formylbicyclomycin (FB) to 3.15, 3.05, and 3.15 A resolution, respectively. The structures show that BCM and its derivatives are nonnucleotide inhibitors that interact with Rho at a pocket adjacent to the ATP and RNA binding sites in the C-terminal half of the protein. BCM association prevents ATP turnover by an unexpected mechanism, occluding the binding of the nucleophilic water molecule required for ATP hydrolysis. Our data explain why only certain elements of BCM have been amenable to modification and serve as a template for the design of new inhibitors.

Adenosine Triphosphatases↗

PubMed2005

The molecular basis for the mode of action of bicyclomycin.

Bicyclomycin (1) is a clinically useful antibiotic exhibiting activity against a broad spectrum of Gram-negative bacteria and against the Gram-positive bacterium, Micrococcus luteus. Bicyclomycin has been used to treat diarrhea in humans and bacterial diarrhea in calves and pigs and is marketed by Fujisawa (Osaka, Japan) under the trade name Bicozamycin. The structure of 1 is unique among antibiotics, and our studies document that its mechanism of action is novel. Early mechanistic proposals suggested that 1 reacted with nucleophiles (e.g., a protein sulfhydryl group) necessary for the remodeling the peptidoglycan assembly within the bacterial cell wall. We, however, showed that 1 targeted the rho transcription termination factor in Escherichia coli. The rho protein is integral to the expression of many gene products in E. coli and other Gram-negative bacteria, and without rho the cell losses viability. Rho is a member of the RecA-type ATPase class of enzymes that use nucleotide contacts to couple oligonucleotide translocation to ATP hydrolysis. Bicyclomycin is the only known selective inhibitor of rho. In this article, we integrate the evidence obtained from bicyclomycin structure-activity studies, site-directed mutagenesis investigations, bicyclomycin affinity labels, and biochemical and biophysical measurements with recent X-ray crystallographic images of the bicyclomycin-rho complex to define the rho antibiotic binding site and to document the pathway for rho inhibition by 1. Together, the structural and functional studies demonstrate how 1, a modest rho inhibitor, can disrupt the rho molecular machinery thereby leading to a catastrophic effect caused by the untimely overproduction of proteins not normally expressed constitutively, thus leading to a toxic effect on the cells.

Adenosine Triphosphatases↗

PubMed2004

N-Substituted amino acid N'-benzylamides: synthesis, anticonvulsant, and metabolic activities.

Amino acid amides (AAA) were prepared and evaluated in seizure models. The AAA displayed moderate-to-excellent activity in the maximal electroshock seizure (MES) test and were devoid of activity in the subcutaneous Metrazol-induced (scMet) seizure test. The AAA anticonvulsant activity was neither strongly influenced by the C(2) substituent nor by the degree of terminal amine substitution. An in vitro metabolism study suggested that the structure-activity relationship pattern was due, in part, to metabolic processes that occurred at the N-terminal amine unit.

Amides↗

PubMed2004

C(8)-substituted 1-azabicyclo[3.3.1]non-3-enes: a novel scaffold for muscarinic receptor ligands.

The [3.3.1]-bicyclic amine, exo-8-benzyloxymethyl-3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-ene (1), has been shown to be a potent competitive antagonist against the hM(1)-hM(5) muscarinic receptors. This heterocyclic system has not been extensively evaluated despite the notable activities reported for other bicyclic amines. Synthetic strategies permitted the selective alteration of five structural sites in 1. Pharmacological evaluation demonstrated that modification of either the C(3) alkoxycarbonyl or the C(4) enol units in 1 gave compounds with high affinity for the hM(1)-hM(5) muscarinic receptors with selectivity for the hM(2) receptor.

Animals↗

PubMed2004

Application of predictive QSAR models to database mining: identification and experimental validation of novel anticonvulsant compounds.

We have developed a drug discovery strategy that employs variable selection quantitative structure-activity relationship (QSAR) models for chemical database mining. The approach starts with the development of rigorously validated QSAR models obtained with the variable selection k nearest neighbor (kNN) method (or, in principle, with any other robust model-building technique). Model validation is based on several statistical criteria, including the randomization of the target property (Y-randomization), independent assessment of the training set model's predictive power using external test sets, and the establishment of the model's applicability domain. All successful models are employed in database mining concurrently; in each case, only variables selected as a result of model building (termed descriptor pharmacophore) are used in chemical similarity searches comparing active compounds of the training set (queries) with those in chemical databases. Specific biological activity (characteristic of the training set compounds) of external database entries found to be within a predefined similarity threshold of the training set molecules is predicted on the basis of the validated QSAR models using the applicability domain criteria. Compounds judged to have high predicted activities by all or the majority of all models are considered as consensus hits. We report on the application of this computational strategy for the first time for the discovery of anticonvulsant agents in the Maybridge and National Cancer Institute (NCI) databases containing ca. 250,000 compounds combined. Forty-eight anticonvulsant agents of the functionalized amino acid (FAA) series were used to build kNN variable selection QSAR models. The 10 best models were applied to mining chemical databases, and 22 compounds were selected as consensus hits. Nine compounds were synthesized and tested at the NIH Epilepsy Branch, Rockville, MD using the same biological test that was employed to assess the anticonvulsant activity of the training set compounds; of these nine, four were exact database hits and five were derived from the hits by minor chemical modifications. Seven of these nine compounds were confirmed to be active, indicating an exceptionally high hit rate. The approach described in this report can be used as a general rational drug discovery tool.

Amides↗

PubMed2004

Cyclic disulfide C8 iminoporfiromycin: nucleophilic activation of a porfiromycin.

The clinical success of mitomycin C (1) and its associated toxicities and resistance have led to efforts to prepare semisynthetic analogues (i.e., KW-2149 (3), BMS-181174 (4)) that have improved pharmacological profiles. In this study, we report the preparation and evaluation of the novel 7-N-(1'-amino-4',5'-dithian-2'-yl)porfiromycin C(8) cyclized imine (6) and its reference compound, 7-N-(1'-aminocyclohex-2'-yl)porfiromycin C(8) cyclized imine (13). Porfiromycin 6 contains a disulfide unit that, upon cleavage, may provide thiol(s) that affect drug reactivity. We demonstrated that phosphines dramatically accelerated 6 activation and solvolysis in methanolic solutions ("pH 7.4") compared with 13. Porfiromycins 6 and 13 efficiently cross-linked EcoRI-linearized pBR322 DNA upon addition of Et3P. We found enhanced levels of interstrand cross-link (ISC) adducts for 6 and 13 compared with porfiromycin (7) and that 6 was more efficient than 13. The large Et3P-mediated rate enhancements for the solvolysis of 6 compared with 13 and a N(7)-substituted analogue of 1, and the increased levels of ISC adducts for 6 compared with 13 and 7 are attributed to a nucleophile-assisted disulfide cleavage process that permits porfiromycin activation and nucleophile (MeOH, DNA) adduction. The in vitro antiproliferative activities of 6 and 13 using the A549 tumor cell line (lung adenocarcinoma) were determined under aerobic and hypoxic conditions and then compared with 7. Both 6 and 13 were more cytotoxic than 7, with 13 being more potent than 6. The C(8) iminoporfiromycins 6 and 13 displayed anticancer profiles similar to 3.

Animals↗

PubMed2003

Synthetic enantiopure aziridinomitosenes: preparation, reactivity, and DNA alkylation studies.

An enantiocontrolled route to aziridinomitosenes had been developed from l-serine methyl ester hydrochloride. The tetracyclic target ring system was assembled by an internal azomethine ylide cycloaddition reaction based on silver ion-assisted intramolecular oxazole alkylation and cyanide-induced ylide generation via a labile oxazoline intermediate (62 to 66). Other key steps include reductive detritylation of 26, methylation of the N-H aziridine 56, oxidation of the sensitive cyclohexenedione 68 to quinone 70, and carbamoylation using Fmoc-NCO. Although the aziridinomitosene tetracycle is sensitive, a range of protecting group manipulations and redox chemistry can be performed if suitable precautions are taken. A study of DNA alkylation by the first C-6,C-7-unsubstituted aziridinomitosene 11a has been carried out, and evidence for DNA cross-link formation involving nucleophilic addition to the quinone subunit is described.

Alkylation↗

PubMed2003

Functionalized amido ketones: new anticonvulsant agents.

We have reported that functionalized amino acids (FAA) are potent anticonvulsants. Replacing the N-terminal amide group in FAA with phenethyl, styryl, and phenylethynyl units provided a series of functionalized amido ketones (FAK). We show that select FAK exhibit significant anticonvulsant activities thereby providing information about the structural requirements for FAA and FAK bioactivity.

Amides↗

PubMed2003

Metal-1,4-dithio-2,3-dihydroxybutane chelates: novel inhibitors of the Rho transcription termination factor.

Rho is an enzyme that is essential for the growth and survival of Escherichia coli, and bicyclomycin (1) is its only known selective inhibitor. We show that metal (Cd(2+), Ni(2+), and Zn(2+)) complexes of 1,4-dithio-2,3-dihydroxybutanes (2) serve as effective and potent rho inhibitors with I(50) values that can exceed that of 1. Maximal inhibition for ZnCl(2) and L-dithiothreitol (2a) corresponded to Zn(2):L-DTT stoichiometry. The I(50) value for the 2:1 Zn-L-DTT solution was 20 microM, which made it 3 times more potent than 1 (I(50) = 60 microM). Kinetic studies showed that a Zn-L-DTT solution functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay and as a competitive inhibitor with respect to ribo(C)(10) in the poly(dC).ribo(C)(10)-stimulated ATPase assay. These findings demonstrated that both 1 and a Zn-L-DTT solution disrupted rho-mediated ATP hydrolysis but that they inhibit using different mechanisms. Substitution of L-DTT with 1,2-ethanedithiol in ZnCl(2) solutions led to a comparable loss of rho poly(C)-dependent ATPase activity, indicating that other metal chelates can serve as efficient inhibitors. The site and pathway of rho inhibition by the putative metal-1,4-dithio-2,3-dihydroxybutane chelates are discussed in light of the current data.

Adenosine Triphosphatases↗

PubMed2003

Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties.

Bicyclomycin (1) is a commercially available antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. Key aspects of the 1.rho interaction-K(d), stoichiometry for 1.rho binding, and whether 1 and ATP binding induce conformational changes in rho-remain unknown. In this study, the design, synthesis, and characterization of a series of bicyclomycin fluorescent probes (BFP) constructed to sense the 1.rho interaction are described and their use documented. We show that dihydrobicyclomycins with medium-to-large C(5a)-substituents afforded excellent inhibitory activities exceeding those of 1 in the poly(C)-dependent ATPase assay. The utility of BFP in bicyclomycin-rho binding studies was documented through the use of 5a-(phenazin-2-ylmethylsulfanyl)dihydrobicyclomycin (15). Excitation (290 nm) of W381 in wild-type rho in the presence of 15 and ATP led to fluorescence resonance energy transfer (FRET) and gave a K(d) (15) of 9.9 microM. Using ADP in place of ATP or excluding nucleotide did not result in energy transfer, which suggests that ATP binding induced a conformational change in rho. FRET measurements provided an approximate weighted average distance (23 A) between W381 and 15 in the presence of bound ATP. The K(d) value for 15.rho was correlated with ATP binding at the 3 tight ATP binding (K(d)(ATP) = 95 nM) sites in wild-type rho.

Adenosine Triphosphate↗

PubMed2003

C(8) substituted 1-azabicyclo[3.3.1]non-3-enes and C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones: novel muscarinic receptor antagonists.

Expedient syntheses of C(8) substituted 1-azabicyclo[3.3.1]non-3-enes and C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones are reported to begin with 2,5-disubstituted pyridines. Catalytic reduction of the pyridine to the piperidine followed by treatment with ethyl acrylate and Dieckmann cyclization gave diastereomeric mixtures of C(8) substituted 3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-enes, which were separable by chromatography. We found that the catalytic reduction (PtO2, H2) procedure provided the cis-substituted piperidine but that pyridine reduction was accompanied by competitive cleavage of the C(2) pyridyl substituent. Accordingly, an alternative route was devised that afforded a diastereomeric mixture of the cis- and trans-2,5-disubstituted piperidine. Treatment of the substituted pyridine with m-CPBA gave the pyridine N-oxide, which was reduced to the piperidine by sequential reduction with ammonium formate in the presence of Pd-C followed by NaBH3CN. Addition of ethyl acrylate completed the synthesis of the substituted piperidine. The overall four-step reaction gave higher yields (57%) than the two-step procedure (13%) with little cleavage of the C(2) pyridyl substituent. Acid decarboxylation of the bicyclo[3.3.1]non-3-enes provided the C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones. Structural studies revealed diagnostic 13C NMR signals that permit assignment of the orientation of the C(8) substituent. Pharmacological investigations documented that 3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-enes efficiently bind to the human M1-M5 muscarinic receptors and function as antagonists. We observed that exo-8-benzyloxymethyl-3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-ene (3) displayed the highest affinity, exhibiting Ki values at all five muscarinic receptors that were approximately 10-50 times lower than carbachol and approximately 30-230 times lower than arecoline. Receptor selectivity was observed for 3. Compound 3 contained two different pharmacophores found in many muscarinic receptor ligands, and preliminary findings indicated the importance of both structural elements for maximal activity. Compound 3 serves as a novel lead compound for further drug development.

Animals↗

PubMed2003

Metal dependency for transcription factor rho activation.

The Escherichia coli rho transcription termination factor terminates select transcripts and rho activity requires Mg(2+). We investigated whether divalent metal ions other than Mg(2+) catalyze rho-dependent ATP hydrolysis to ADP and P(i) in vitro. The effects of 11 divalent metal ions (Be(2+), Ca(2+), Cd(2+), Co(2+), Cu(2+), Hg(2+), Mn(2+), Ni(2+), Sr(2+), VO(2+), Zn(2+)) on ATPase activity were determined in the absence and presence of MgCl(2). Without MgCl(2), Ca(2+), Cd(2+), Co(2+), Cu(2+), Hg(2+), Mn(2+), Ni(2+), VO(2+), and Zn(2+) activated ATP hydrolysis with either hyberbolic (Ca(2+), Co(2+), Cu(2+), Hg(2+), VO(2+)), peak velocity (Cd(2+), Mn(2+), Zn(2+)), or sigmoidal (Ni(2+)) rate acceleration curves. Sr(2+) was found to be a nonactivator and Be(2+) an inhibitor of rho-dependent ATPase activity. The metals' effects were compared with Mg(2+) and gave different rank orders when either the velocity (V(max), V(peak)) or the efficiency (V(max)/K(M), V(peak)/K(M)) of ATP hydrolysis was used as the determinant (V: Mg(2+) approximately Mn(2+) > Zn(2+) > Co(2+) > Ni(2+) approximately Cd(2+) > Ca(2+) > Cu(2+) > Hg(2+) approximately VO(2+); V/K(M): Mg(2+) > Mn(2+) > Ca(2+) > Co(2+) > Zn(2+) > Cu(2+) > Ni(2+) > Hg(2+) > Cd(2+)). Mg(2+) proved to be the most effective divalent metal. We observed that the metal-dependent rates were affected by metal ion interactions with rho, RNA, and the buffer constituents. Significantly, replacement of the octahedral Mg(2+) ion by metals that typically prefer coordination spheres less than six (Cd(2+), Co(2+), Ni(2+), VO(2+), Zn(2+)) led to ATPase activity, suggesting that the putative Mg x ATP(2-) coordination sphere in rho does not need to remain fully intact for ATP hydrolysis.

Adenosine Triphosphate↗