Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs

Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the beta-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182-->Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.     

Formation of aminoxyl radicals in the reaction between penicillins and hydrogen peroxide.

Aminoxyl radicals are formed in high yield in the reaction between penicillins and hydrogen peroxide in water solutions in the pH range between 7 and 8. The nine-line EPR spectrum, 3 x 3 (1:2:1), indicated an interaction of the unpaired electron with one 14N nucleus (aN = 1.44 mT) and two equivalent hydrogen nuclei (aH = 2.00 mT). The reaction involves an oxidative cleavage of the beta-lactam ring of the penicillins with the formation of a cyclic aminoxyl radical, in which the thiazolidine ring carries the nitroxide group (= N-O.). It is suggested that the reaction with the formation of aminoxyl radicals can also take place in vivo in the deactivation of penicillins by metabolically formed hydrogen peroxide.     

Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1

Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.     

Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.     

Structure-activity relations and beta-lactamase resistance

beta-Lactam antibiotics resistant to beta-lactamase degradation can be produced by many chemical modifications, but often at the expense of antibacterial activity. Substitution onto several positions in the molecule produces different and often selective resistance; for instance, heavily sterically hindered acyl groups give staphylococcal beta-lactamase resistance to penicillins, and resistance to some enzymes from Gram-negative pathogens to both penicillins and cephalosporins. 6-alpha- or 7-alpha-substituents respectively confer a broad spectrum of resistance (e.g. cefoxitin), but changes at positions 2 or 3 have only a minor influence on enzyme susceptibility. Changes in the ring condensed with the beta-lactam, such as changing ceph-3-em to ceph-2-em may greatly enhance stability. Small improvement can occur when the nuclear sulphur atom is oxidized, but a much better effect is obtained when it is replaced by another atom such as oxygen, as in clavulanic acid. This compound appears to have broad spectrum resistance which is actually due to susceptibility and subsequent produce inhibition.     

Natural Variants of the KPC-2 Carbapenemase have Evolved Increased Catalytic Efficiency for Ceftazidime Hydrolysis at the Cost of Enzyme Stability

The spread of β-lactamases that hydrolyze penicillins, cephalosporins and carbapenems among Gram-negative bacteria has limited options for treating bacterial infections. Initially, Klebsiella pneumoniae carbapenemase-2 (KPC-2) emerged as a widespread carbapenem hydrolyzing β-lactamase that also hydrolyzes penicillins and cephalosporins but not cephamycins and ceftazidime. In recent years, single and double amino acid substitution variants of KPC-2 have emerged among clinical isolates that show increased resistance to ceftazidime. Because it confers multi-drug resistance, KPC β-lactamase is a threat to public health. In this study, the evolution of KPC-2 function was determined in nine clinically isolated variants by examining the effects of the substitutions on enzyme kinetic parameters, protein stability and antibiotic resistance profile. The results indicate that the amino acid substitutions associated with KPC-2 natural variants lead to increased catalytic efficiency for ceftazidime hydrolysis and a consequent increase in ceftazidime resistance. Single substitutions lead to modest increases in catalytic activity while the double mutants exhibit significantly increased ceftazidime hydrolysis and resistance levels. The P104R, V240G and H274Y substitutions in single and double mutant combinations lead to the largest increases in ceftazidime hydrolysis and resistance. Molecular modeling suggests that the P104R and H274Y mutations could facilitate ceftazidime hydrolysis through increased hydrogen bonding interactions with the substrate while the V240G substitution may enhance backbone flexibility so that larger substrates might be accommodated in the active site. Additionally, we observed a strong correlation between gain of catalytic function for ceftazidime hydrolysis and loss of enzyme stability, which is in agreement with the ‘stability-function tradeoff’ phenomenon. The high T_m of KPC-2 (66.5°C) provides an evolutionary advantage as compared to other class A enzymes such as TEM (51.5°C) and CTX-M (51°C) in that it can acquire multiple destabilizing substitutions without losing the ability to fold into a functional enzyme.     

Relationship between the antibacterial activity and the acceptor properties of certain penicillins]

Antibacterial activity of 2 natural and 12 semisynthetic penicillins against 5 strains of grampositive bacteria was determined and quantum chemical estimation of their molecules was performed with the Hukkel method. The data were indicative of the fact that antibacterial activity of the penicillins was connected with the acceptor properties of their molecules.     

ATP-analogues as substrates for the leucyl-tRNA synthetase from Escherichia coli MRE 600.

No analogous nucleoside triphosphate was found which acts as well as ATP in binding to and supporting catalysis of leucyl-tRNA synthetase from Escherichia coli MRE 600. However, there are numerous nucleotides which are able to replace ATP, but with lower efficiency. The 6-amino group of the adenine ring and the 2'-hydroxyl group of the ribose ring are essential for binding and catalytic activity. Alterations in the triphosphate moiety of the molecule can cause drastic changes in Km and/or Vmax, whereas alterations of the imidazole ring and substitutions at the 8-position of the adenine ring cause only minor losses of catalytic activity.     

Theoretical studies on penicillins,penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues: binding specificities of transeptidases and penicillinases

Empirical potential energy calculations have been carried out to determine the preferred conformations of penicillins and penicillin sulphones and their 1-oxa-1-dethia and 1-carba-1-dethia analogues. With the exception of 1-oxa-1-dethia penicillins, all the other compounds favour C₂ and the C₃ puckered conformations of their five-membered rings. Replacement of C2 methyl groups by hydrogen atoms as in bisnorpenicillin V or oxidation of sulphur in position 1 as in sulphones, makes the C₃ puckered form much less favourable. Addition of an amino-acyl group at the C6 atom, however, makes the C₃ puckered form more favoured in penicillin G or V and in 1-carba-1-dethia penicillins. Through the replacement of the sulphur atom at position 1 by an oxygen atom or by a -CH₂ group increases the non-planarity of the lactam peptide bond, it significantly affects the relative disposition of the C3 carboxyl group with respect to the β-lactam ring. These conformational differences have been correlated with the biological activities of these compounds. The present study suggests that the conformation of the bicyclic ring system may be more important for initial binding with the crosslinking enzyme(s) involved in the biosynthesis of bacterial cell-wall peptidoglycan and that the mode of binding is influenced by the nature of the side-group at the C6 atom. These studies predict, in agreement with experimental results, that the 1-oxa-1-dethia penicillin nulceus is an inhibitor of penicillianses. The study also suggests that the stereospecificities of the crosslinking enzyme(s) and penicillinases are very similar with regard to the nature of the side-group at the 6 atom and the confirmation of the bicyclic ring system. However, the confirmational requirement for the bicyclic ring system appears to be more specific in the former enzyme than in the latter.     

Synthesis and Antiviral Activity of 2-Substituted Analogs of Triciribine

Abstract

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore the effect of substitutions at the 2-position of triciribine. 2-Methyl-(2-Me-TCN), 2-ethyl-(2-Et-TCN), 2-phenyl-(2-Ph-TCN), 2-chloro-(2-Cl-TCN), and 2-aminotriciribine(2-NH₂-TCN) were designed and synthesized to determine the effects of substitutions at the 2-position which change the steric, electronic, and hydrophobic properties of TCN, while maintaining the integrity of the tricyclic ring system. These compounds were evaluated for activity against human immunodeficiency virus (HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV) and were found to be either less active than TCN and TCN-P or inactive at the highest concentrations tested, 100 µM. We conclude that substitutions at the 2-position of triciribine adversely affect the antiviral activity most likely because these analogs are not phosphorylated to active metabolites.     

Synthesis, in silico metabolic and toxicity prediction of some novel imidazolinones derivatives as potent anticonvulsant agents

A series of 1,2,4-trisubstituted 5-imidazolinone derivatives were synthesized by Erlenmeyer condensation of benzoylglycine (hippuric acid) with different aldehydes in the presence of sodium acetate and acetic anhydride. The derivatives of the compounds were prepared by condensation of some known sulpha drugs with 5-oxazolone derivatives. The anticonvulsant activity of the compounds was determined by the protection of pentylenetetrazole-induced convulsions that was ranged from 10 to 60%. The compounds with p-OCH?, p-OH and o-Cl substitutions in the phenyl ring on 4(th) position of the imidazolinone ring exhibited good anticonvulsant activity. In silico metabolic and toxicity studies showed that all the compounds in the series are not likely to exhibit toxicity except the compounds IIIa, IIIb, VIa and VIb, that is predicted to show 29% mutagenicity and 53% irritation in comparison to the other compounds. The predicted lethal effect and hERG toxicity of the compounds showed that IIa, IVa, Va and Vb might be toxic at higher concentrations. The results successfully establish the synthesized imidazolinone derivatives as novel compounds with anticonvulsant properties, low predicted cardiotoxicity and lethal effects thus can be promising leads for further development as novel anticonvulsants.     

Structure-activity relationships amongst beta-lactamase inhibitors

Using a variety of beta-lactamases including those from Escherichia coli (TEM-1), Enterobacter cloacae P99 and Staphylococcus aureus the inhibition profiles (I50 values) were determined for various groups of compounds including penicillins, penicillanic acid derivatives (sulphone and beta-halo substitutions), olivanic acids and clavulanic acid derivatives including substituted ethers and amines. Some of the latter compounds had higher activity than clavulanic acid with and without preincubation of enzyme with inhibitor but they still had poor activity against the P99 enzyme. Improvements in activity against Class I cephalosporinases were obtained with some derivatives of clavulanic acid but this was usually achieved at the expense of activity against clavulanate susceptible beta-lactamases. The olivanic acids had the highest activity against the widest range of beta-lactamases.     

Theoretical studies on β-lactam antibiotics. IV. Conformational analysis of novel β-lactam antibiotics and the binding specificities of crosslinking enzyme(s) and β-lactamases

Conformational-energy calculations were carried out on the new family of β-lactam antibiotics (viz., thienamycin, PS-5, 1-oxa- and 1-thiapenems, and their close analogs); these exhibit broad-spectrum antibacterial activity and stability towards β-lactamase-producing strains. The bicyclic ring system in all the compounds studied was found to be highly rigid and to favor only one conformation. This is in contrast to findings in penicillins, where the five-membered ring assumes two puckered conformations. The relative orientations of the bicyclic ring system and the nature and configuration of the substituent at C-5 position, besides nonplanarity of the lactam peptide bond, are shown to be important for biological activity. The present study, in agreement with x-ray studies, predicts that the lactam peptide bond in 1-carbapenem is more nonplanar than in 1-thiapenem. These studies also suggest that the conformational requirement of bicyclic ring system to bind to crosslinking enzyme(s) and β-lactamases is very similar.     

Conservation of physico-chemical amino acid properties during the evolution of proteins

Based on a similarity ring constructed from a substitution probability matrix, we have analyzed the conservation of some amino acid properties in the evolution of proteins. Refractive index and bulkiness are highly conserved, hydrophobicity and polarity are fairly well conserved while optical rotation appears to be a less relevant property. On the other hand, the analysis of the correspondence between phenotype and genotype shows that the most frequent amino acid substitutions in proteins do not always correspond to the most feasible codon changes. The apparent disagreement between amino acid substitutions in modern proteins and the primordial amino acid-codon assignment is discussed.     

Structural and kinetic studies on beta-lactamase K1 from Klebsiella aerogenes.

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.     

The preference of tryptophan for membrane interfaces: insights from N-methylation of tryptophans in gramicidin channels

To better understand the structural and functional roles of tryptophan at the membrane/water interface in membrane proteins, we examined the structural and functional consequences of Trp --> 1-methyl-tryptophan substitutions in membrane-spanning gramicidin A channels. Gramicidin A channels are miniproteins that are anchored to the interface by four Trps near the C terminus of each subunit in a membrane-spanning dimer. We masked the hydrogen bonding ability of individual or multiple Trps by 1-methylation of the indole ring and examined the structural and functional changes using circular dichroism spectroscopy, size exclusion chromatography, solid state (2)H NMR spectroscopy, and single channel analysis. N-Methylation causes distinct changes in the subunit conformational preference, channel-forming propensity, single channel conductance and lifetime, and average indole ring orientations within the membrane-spanning channels. The extent of the local ring dynamic wobble does not increase, and may decrease slightly, when the indole NH is replaced by the non-hydrogen-bonding and more bulky and hydrophobic N-CH(3) group. The changes in conformational preference, which are associated with a shift in the distribution of the aromatic residues across the bilayer, are similar to those observed previously with Trp --> Phe substitutions. We conclude that indole N-H hydrogen bonding is of major importance for the folding of gramicidin channels. The changes in ion permeability, however, are quite different for Trp --> Phe and Trp --> 1-methyl-tryptophan substitutions, indicating that the indole dipole moment and perhaps also ring size and are important for ion permeation through these channels.     

Synthesis and antiviral activity of 2-substituted analogs of triciribine

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore the effect of substitutions at the 2-position of triciribine. 2-Methyl- (2-Me-TCN), 2-ethyl- (2-Et-TCN), 2-phenyl- (2-Ph-TCN), 2-chloro- (2-Cl-TCN), and 2-aminotriciribine (2-NH2-TCN) were designed and synthesized to determine the effects of substitutions at the 2-position which change the steric, electronic, and hydrophobic properties of TCN, while maintaining the integrity of the tricyclic ring system. These compounds were evaluated for activity against human immunodeficiency virus (HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV) and were found to be either less active than TCN and TCN-P or inactive at the highest concentrations tested, 100 microM. We conclude that substitutions at the 2-position of triciribine adversely affect the antiviral activity most likely because these analogs are not phosphorylated to active metabolites.     

Functionalization of the A ring of pyridoacridine as a route toward greater structural diversity. Synthesis of an octacyclic analogue of eilatin

In an effort to increase the structural diversity of pyrido[4,3,2-kl]acridines, compounds containing amino substituents on the A ring were synthesized. The key-reactions involve regioselective electrophilic aromatic substitutions. The methodology was applied to the synthesis of the extended angular octacycle 8, which conjugates the physicochemical and spectroscopic properties of the pyridoacridine skeleton with the ability of [1,10]phenanthroline ring for metal complexation. The 9-aminopyridoacridine 4 displays significant cytostatic activities against two cancer cell lines, and may be considered as a new lead in the search of active derivatives.     

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.     

Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.