scholarly journals Interactive design and synthesis of a novel antibacterial agent

1994 ◽  
Vol 72 (4) ◽  
pp. 1051-1065 ◽  
Author(s):  
Saul Wolfe ◽  
Haolun Jin ◽  
Kiyull Yang ◽  
Chan-Kyung Kim ◽  
Ernest McEachern
Keyword(s):  
Molecular Orbital ◽  
Active Site ◽  
Antibacterial Agent ◽  
De Novo ◽  
Three Dimensional ◽  
Hydroxyl Group ◽  
Molecular Orbital Calculations ◽  
Binding Studies ◽  
Molecular Mechanics Calculations ◽  
Substrate Complex

β-Lactam compounds act on penicillin-recognizing enzymes via acylation of the hydroxyl group of an active site serine. When the resulting acyl enzyme is kinetically stable, as in the case of a penicillin-binding protein (PBP), the biosynthesis of a bacterial cell wall is inhibited, and death of the organism results. The de novo design of an antibacterial agent targeted to a PBP might be possible if the three-dimensional structural requirements of the equilibrium (i.e, fit) and catalytic (i.e. reactivity) steps of the aforementioned enzymatic process could be determined. For a model of the active site of a PBP from Streptomyces R61, the use of molecular mechanics calculations to treat "fit," and ab initio molecular orbital calculations to treat "reactivity," leads to the idea that the carboxyl group (G1) and the amide N-H (G2) of the antibiotic are hydrogen bonded to a lysine amino group and a valine carbonyl group in the enzyme–substrate complex. These two hydrogen bonds place the serine hydroxyl group on the convex face of the antibiotic, in position for attack on the β-lactam ring by a neutral reaction, catalyzed by water, that involves a direct proton transfer to the β-lactam nitrogen. Molecular orbital calculations of structure–reactivity relations associated with this mechanism suggest that C=N is bioisosteric to the β-lactam N-C(=O), comparable to a β-lactam in its reactivity with an alcohol, and that the product RO(C-N)H is formed essentially irreversibly (−ΔE > 10 kcal/mol). Accordingly, structures containing a G1 and a G2 separated by a C=N, and positioned in different ways with respect to this functional group, have been synthesized computationally and examined for their ability to fit to the PBP model. This strategy identified a 2H-5,6-dihydro-1,4-thiazine substituted by hydroxyl and carboxyl groups as a target for chemical synthesis. However, exploratory experiments suggested that the C=N of this compound equilibrates with endocyclic and exocyclic enamine tautomers. This required that the C2 position be substituted, and that the hydroxyl group not be attached to the carbon atom adjacent to the C=N. These conditions are met in a 2,2-dimethyl-3-(2-hydroxypropyl)-1,4-thiazine, which also exhibits the necessary fit to the PBP model. Two epimers of this compound have been synthesized, from D- and L-serine. The compound derived from L-serine is not active. The compound derived from D-serine exhibits antibacterial activity, but is unstable, and binding studies with PBP's have not been performed. It is hoped that these studies can be carried out if modification of the lead structure leads to compounds with improved chemical stability.

scholarly journals Structural insights into enzymatic [4+2] aza-cycloaddition in thiopeptide antibiotic biosynthesis

2017 ◽  
Vol 114 (49) ◽  
pp. 12928-12933 ◽  
Author(s):  
Dillon P. Cogan ◽  
Graham A. Hudson ◽  
Zhengan Zhang ◽  
Taras V. Pogorelov ◽  
Wilfred A. van der Donk ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Active Sites ◽  
De Novo ◽  
Core Protein ◽  
Cycloaddition Reaction ◽  
Structural Similarity ◽  
Antibiotic Biosynthesis ◽  
Binding Studies ◽  
Nitrogenous Heterocycle

The [4+2] cycloaddition reaction is an enabling transformation in modern synthetic organic chemistry, but there are only limited examples of dedicated natural enzymes that can catalyze this transformation. Thiopeptides (or more formally thiazolyl peptides) are a class of thiazole-containing, highly modified, macrocyclic secondary metabolites made from ribosomally synthesized precursor peptides. The characteristic feature of these natural products is a six-membered nitrogenous heterocycle that is assembled via a formal [4+2] cycloaddition between two dehydroalanine (Dha) residues. This heteroannulation is entirely contingent on enzyme activity, although the mechanism of the requisite pyridine/dehydropiperidine synthase remains to be elucidated. The unusual aza-cylic product is distinct from the more common carbocyclic products of synthetic and biosynthetic [4+2] cycloaddition reactions. To elucidate the mechanism of cycloaddition, we have determined atomic resolution structures of the pyridine synthases involved in the biosynthesis of the thiopeptides thiomuracin (TbtD) and GE2270A (PbtD), in complex with substrates and product analogs. Structure-guided biochemical, mutational, computational, and binding studies elucidate active-site features that explain how orthologs can generate rigid macrocyclic scaffolds of different sizes. Notably, the pyridine synthases show structural similarity to the elimination domain of lanthipeptide dehydratases, wherein insertions of secondary structural elements result in the formation of a distinct active site that catalyzes different chemistry. Comparative analysis identifies other catalysts that contain a shared core protein fold but whose active sites are located in entirely different regions, illustrating a principle predicted from efforts in de novo protein design.

Molecular mechanism of chromogenic substrate hydrolysis in the active site of human carboxylesterase-1

2021 ◽  
Vol 67 (3) ◽  
pp. 300-305
Author(s):  
A.M. Kulakova ◽  
M.G. Khrenova ◽  
A.V. Nemukhin
Keyword(s):  
Molecular Mechanism ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Kinetic Equations ◽  
Binding Constants ◽  
Forward Direction ◽  
Rational Drug Design ◽  
Molecular Mechanics Calculations ◽  
Elementary Steps ◽  
Substrate Complex

Human carboxylesterases are involved in the protective processes of detoxification during the hydrolytic metabolism of xenobiotics. Knowledge of the molecular mechanisms of substrates hydrolysis in the enzymes active site is necessary for the rational drug design. In this work, the molecular mechanism of the hydrolysis reaction of para-nitrophenyl acetate in the active site of human carboxylesterase was determined using modern methods of molecular modeling. According to the combined method of quantum mechanics/molecular mechanics calculations, the chemical reaction occurs within four elementary steps, including two steps of the acylation stage, and two steps of the deacylation stage. All elementary steps have low energy barriers, with the gradual lowering of the intermediate energies that stimulates reaction in the forward direction. The molecular docking was used to estimate the binding constants of the enzyme-substrate complex and the dissociation constant of enzyme-product complexes. The effective kinetic parameters of the enzymatic hydrolysis in the active site of carboxylesterase are determined by numerical solution of the differential kinetic equations.

scholarly journals Proposal of Potent Inhibitors for a Bacterial Cell Division Protein FtsZ: Molecular Simulations Based on Molecular Docking and ab Initio Molecular Orbital Calculations

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 846
Author(s):  
Shohei Yamamoto ◽  
Ryosuke Saito ◽  
Shunya Nakamura ◽  
Haruki Sogawa ◽  
Pavel Karpov ◽  
...  
Keyword(s):  
Cell Division ◽  
Ab Initio ◽  
Molecular Orbital ◽  
Bacterial Cell ◽  
Molecular Simulations ◽  
Ligand Docking ◽  
Hydroxyl Group ◽  
Molecular Orbital Calculations ◽  
Bacterial Cell Division ◽  
Cell Division Protein

The inhibition of a bacterial cell division protein, filamentous temperature-sensitive Z (FtsZ), prevents the reproduction of Mycobacteria. To propose potent inhibitors of FtsZ, the binding properties of FtsZ with various derivatives of Zantrin ZZ3 were investigated at an electronic level, using molecular simulations. We here employed protein–ligand docking, classical molecular mechanics (MM) optimizations, and ab initio fragment molecular orbital (FMO) calculations. Based on the specific interactions between FtsZ and the derivatives, as determined by FMO calculations, we proposed novel ligands, which can strongly bind to FtsZ and inhibit its aggregations. The introduction of a hydroxyl group into ZZ3 was found to enhance its binding affinity to FtsZ.

Molecular Orbital Studies of Enzyme Catalysed Reactions. Rearrangement of Chorismate to Prephenate

1979 ◽  
Vol 32 (9) ◽  
pp. 1921 ◽  
Author(s):  
PR Andrews ◽  
RC Haddon
Keyword(s):  
Aqueous Solution ◽  
Transition State ◽  
Molecular Orbital ◽  
Active Site ◽  
Active Sites ◽  
Transition States ◽  
Reaction Pathways ◽  
Carboxyl Groups ◽  
Molecular Orbital Calculations ◽  
Charge Delocalization

Molecular orbital calculations are used to describe the reaction surface for the non-enzymic Claisen rearrangement of chorismate to prephenate, which may proceed through either a boat-like or a chair-like transition state. Detailed molecular geometries are obtained for the neutral and dianionic forms of chorismate, prephenate, and the alternative transition states. The transition states are asymmetric structures in which the breaking C-O bond (c. 1.45 A) is significantly shorter than the making C-C bond (c. 1.95 A). The alternative reaction pathways have almost identical enthalpies of activation (chair, 277.4 kJ/mol ; boat, 282.8 kJ/mol; dianionic forms) which result partly from a loss of internal bond strength and partly from repulsive interactions between the polar carboxyl groups. Protonation stabilizes the transition states (chair, 247.3 kJ/mol; boat, 248.5 kJ/mol ; diacid forms) by delocalization of charge in the carboxyl groups, and a similar mechanism is proposed for the greatly reduced enthalpy of activation in aqueous solution (86.6 kJ/mol). The enthalpy difference between the alternative reaction pathways is insufficient to define a preferred transition state structure, and either pathway may be favoured for the non-enzymic reaction in aqueous solution. For the enzyme-catalysed reaction the chair pathway is used, and the calculated transition state structures and enthalpy barriers provide information relevant to the catalytic mechanism. They indicate that an active site comprising only two essential binding groups is sufficient to account for catalysis; the orientation of these groups within the active site should allow simultaneous bond formation, accompanied by charge delocalization, to both carboxyl groups of the transition state, but not to those of substrate or product. The calculated structure for the chair transition state, taken in conjunction with those for chorismate and prephenate, thus provides a template for the active sites of chorismate mutases.

Synthesis of the stereoisomers of a novel antibacterial agent and interpretation of their relative activities in terms of a theoretical model of the penicillin receptor

1994 ◽  
Vol 72 (4) ◽  
pp. 1066-1075 ◽  
Author(s):  
Saul Wolfe ◽  
Caijun Zhang ◽  
Blair D. Johnston ◽  
Chan-Kyung Kim
Keyword(s):  
Theoretical Model ◽  
Antibacterial Agent ◽  
Hydroxyl Group ◽  
Agent Based ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Chemical Hydrolysis ◽  
Optimum Geometry ◽  
The Stability ◽  
Optically Pure

2,2-Dimethyl-3-(2′-hydroxypropyl)-5-carboxy-Δ3 -1,4-thiazine (1) is a designed antibacterial agent. Based on an analysis of how penicillin complexes to and reacts with a model of a penicillin-binding protein, 1 contains a functional group (C=N) that can react with a serine hydroxyl group of the receptor according to the putative reaction Enz-OH + C = N → Enz-O-C-NH. Compound 1 also contains additional substituents that are designed to position the O-H and C=N groups relative to one another in the enzyme–substrate complex in a geometry that attempts to reproduce the optimum geometry of approach of two such reactants. A most important assumption is that this optimum geometry can be computed ab initio. In a first preparation of 1, (±)-5-methyl-4-hexene-2-ol (2) was converted to the lithium salt of (±)-2-mercapto-2-methyl-5-tert-butyldimethylsiloxy-3- hexanone (7), which was condensed with the N-tert-butoxycarbonyl-D- and L-serine-β-lactones (3). The synthesis was completed by deprotection with formic acid and cyclization in water. The R and S enantiomers of 2 have now been obtained, and the absolute configuration of the alcohol established, by reaction of the R- and S-propylene oxides with an organometallic reagent prepared from β,β-dimethylvinyl bromide. The R alcohol has also been secured by lipase-catalyzed transesterification with trifluoroethyl butyrate, and chemical hydrolysis of the trifluoroethyl ester. The R and S enantiomers of 2 were converted to the R and S enantiomers of 7, and these were condensed with the R and S enantiomers of 3 to yield each of the stereoisomers of the chemically unstable 1 in ca. 95% optically pure form. Antibacterial activity resides in the 5S,8R and 5S,8S isomers. These findings are shown to be consistent with the theoretical model. It is hoped that the stability of the lead structure 1 can be improved, to allow binding experiments with penicillin recognizing enzymes to proceed.

scholarly journals Crystallographic and molecular-orbital studies on the geometry of antifolate drugs

1980 ◽  
Vol 187 (2) ◽  
pp. 533-536 ◽  
Author(s):  
W E Hunt ◽  
C H Schwalbe ◽  
K Bird ◽  
P D Mallinson
Keyword(s):  
Molecular Orbital ◽  
Plane Deformation ◽  
Carbonyl Oxygen ◽  
British Library ◽  
Molecular Orbital Calculations ◽  
Amino Groups ◽  
Binding Studies ◽  
Reductase Inhibitors ◽  
Out Of Plane ◽  
Bonding Properties

In the common dihydrofolate reductase inhibitors an amino substituent replaces the pteridine carbonyl oxygen atom of folates, with altered hydrogen-bonding properties and size. Flexibility in the amino groups could facilitate enzyme binding. Studies of cycloguanil hydrochloride by neutron diffraction show both in-plane and out-of-plane deformation of amino groups. Molecular-orbital calculations ab initio on 2,4-diamino-5-methylpyrimidinium cation confirm that the 4-amino group is readily deformable. The 2,4-diaminoquinazoline structure is reported. Atomic co-ordinates, thermal parameters, bond distances and bond angles for cycloguanil and 2,4-diaminoquinazoline have been deposited as Supplementary Publication SUP 50108 (13 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23, 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

scholarly journals Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

2007 ◽  
Vol 282 (46) ◽  
pp. 33572-33582 ◽  
Author(s):  
Judit Tóth ◽  
Balázs Varga ◽  
Mihály Kovács ◽  
András Málnási-Csizmadia ◽  
Beáta G. Vértessy
Keyword(s):  
Steady State ◽  
Active Site ◽  
Reaction Pathway ◽  
Three Dimensional ◽  
Kinetic Mechanism ◽  
Dimensional Structure ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Novel Target ◽  
Rate Limiting

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize γ-32P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

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