Increased hydrophobic interactions of iclaprim with Staphylococcus aureus dihydrofolate reductase are responsible for the increase in affinity and antibacterial activity

Oefner, Christian; Bandera, M; Haldimann, Andreas; Laue, Heike; Schulz, Henk; Mukhija, Seema; Parisi, Sandro; Weiss, L.; Lociuro, Sergio; Dale, Glenn E.

doi:10.1093/jac/dkp024

Cited by 93 publications

(85 citation statements)

References 38 publications

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“…The IC 50 and K m values were used to compute the equilibrium inhibition constant (K i ) by using the formalism of Cheng-Prusoff (8). (13,14,20,26). Protein production, purification, and crystallization.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

Barrow¹,

Bunce

Bourne³

et al. 2010

Antimicrob Agents Chemother

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The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to Gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin-and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.

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Section: Methodsmentioning

confidence: 99%

“…Other cocrystal structures have recently been determined with saDHFR and inhibitors, including TMP (22), Iclaprim (26,27), and a series of propargyl-containing molecules (19,20). Like RAB1, each of these molecules contains a DAP ring which docks essentially identically in the protein binding site.…”

Section: Nm)mentioning

confidence: 99%

Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

Barrow¹,

Bunce

Bourne³

et al. 2010

Antimicrob Agents Chemother

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“…Comparisons of the Sa(V31L/F98Y) structure with WT Sa/NADPH/DHF (PDB ID code 3FRD) (15) indicate that a shift in the Phe-92 peptide carbonyl would have little or no effect on DHF binding or turnover, because there are no direct interactions between the two groups. Any minor steric interactions between Phe-92 and the pterin ring of DHF may be compensated by the additional interactions in the glutamate tail, which remains undisturbed.…”

Section: Structure Of Sa(v31l/f98y)dhfrmentioning

confidence: 99%

Protein design algorithms predict viable resistance to an experimental antifolate

Reeve

Gainza²,

Frey

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Methods to accurately predict potential drug target mutations in response to early-stage leads could drive the design of more resilient first generation drug candidates. In this study, a structurebased protein design algorithm (K* in the OSPREY suite) was used to prospectively identify single-nucleotide polymorphisms that confer resistance to an experimental inhibitor effective against dihydrofolate reductase (DHFR) from Staphylococcus aureus. Four of the top-ranked mutations in DHFR were found to be catalytically competent and resistant to the inhibitor. Selection of resistant bacteria in vitro reveals that two of the predicted mutations arise in the background of a compensatory mutation. Using enzyme kinetics, microbiology, and crystal structures of the complexes, we determined the fitness of the mutant enzymes and strains, the structural basis of resistance, and the compensatory relationship of the mutations. To our knowledge, this work illustrates the first application of protein design algorithms to prospectively predict viable resistance mutations that arise in bacteria under antibiotic pressure.drug resistance | antifolate | protein design | DHFR | MRSA

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“…We first investigated whether the excess of thymidine in rat serum also could antagonize the bactericidal activity of iclaprim, a more potent DHFR inhibitor than TMP (28). Similarly to that of TMP-SMX, the bactericidal activity of iclaprim against wild-type MRSA AW6 was abolished in standard killing curves in the presence of rat serum.…”

Section: Discussionmentioning

confidence: 99%

“…Iclaprim selectively blocks bacterial dihydrofolate reductase (DHFR), a key enzyme in the bacterial folate pathway, and inhibits the de novo biosynthesis of thymidylate, one of the precursors of DNA (15,31). In addition to its activity against multidrugresistant pathogens, iclaprim possesses advantages in the context of multiresistance: (i) compared to the diaminopyrimidine trimethoprim (TMP), it has a higher affinity for its molecular target, DHFR (28), an increased antibacterial activity, and a lower propensity for the development of resistance (31); (ii) compared to other antibiotic classes currently used in the clinical setting to treat MRSA infections, iclaprim has a differentiated mechanism of action (28) and does not exhibit cross-resistance.…”

mentioning

confidence: 99%

Efficacy of Iclaprim against Wild-Type and Thymidine Kinase-Deficient Methicillin-Resistant Staphylococcus aureus Isolates in an In Vitro Fibrin Clot Model

Entenza

Haldimann²,

Giddey

et al. 2009

Antimicrob Agents Chemother

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Iclaprim is a novel diaminopyrimidine antibiotic that is active against methicillin-resistant Staphylococcus aureus (MRSA). However, it is known that the activity of diaminopyrimidines against S. aureus is antagonized by thymidine through uptake and conversion to thymidylate by thymidine kinase. Unlike with humans, for whom thymidine levels are low, thymidine levels in rodents are high, thus precluding the accurate evaluation of iclaprim efficacy in animal models. We have studied the bactericidal activity of iclaprim against an isogenic pair of MRSA isolates, the wild-type parent AW6 and its thymidine kinase-deficient mutant AH1252, in an in vitro fibrin clot model. Clots, which were aimed at mimicking vegetation structure, were made from human or rat plasma containing either the parent AW6 or the mutant AH1252, and they were exposed to homologous serum supplemented with iclaprim (3.5 g/ml), trimethoprim-sulfamethoxazole (TMP-SMX; 8/40 g/ml), vancomycin (40 g/ml), or saline, each of which was added one time for 48 h. In rat clots, iclaprim and TMP-SMX were bacteriostatic against the parent, AW6. In contrast, they were bactericidal (>3 log 10 CFU/clot killing of the original inoculum) against the mutant AH1252. Vancomycin was the most active drug against AW6 (P < 0.05), but it showed an activity similar those of iclaprim and TMP-SMX against AH1252. In human clots, iclaprim was bactericidal against both AW6 and AH1252 strains and was as effective as TMP-SMX and vancomycin (P > 0.05). Future studies of animals using simulated human kinetics of iclaprim and thymidine kinase-deficient MRSA, which eliminate the thymidine-induced confounding effect, are warranted to support the use of iclaprim in the treatment of severe MRSA infections in humans.

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Increased hydrophobic interactions of iclaprim with Staphylococcus aureus dihydrofolate reductase are responsible for the increase in affinity and antibacterial activity

Cited by 93 publications

References 38 publications

Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

Protein design algorithms predict viable resistance to an experimental antifolate

Efficacy of Iclaprim against Wild-Type and Thymidine Kinase-Deficient Methicillin-Resistant Staphylococcus aureus Isolates in an In Vitro Fibrin Clot Model

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