Antistaphylococcal Activity of Dihydrophthalazine Antifolates, a Family of Novel Antibacterial Drugs

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.

Section: Discussionsupporting

confidence: 71%

“…Concomitant with the current work, the broad-spectrum activities of related RAB1-like molecules have been demonstrated by Basilea Pharmaceutica International AG. These studies also highlighted the difficulty in generating spontaneous resistant mu-tations of chromosomal DHFR to inhibitors in the dihydrophthalazine series (7,9).…”

mentioning

confidence: 99%

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

Barrow¹,

Bunce

Bourne³

et al. 2010

“…Such mechanisms have been described in other bacterial species (56). Selection of linezolid-resistant mutants in S. aureus and E. faecalis strains has been demonstrated previously (12,37,39), and in the present study a similar propensity to select resistant clones was observed. A resistance mechanism to linezolid in S. aureus and E. faecalis has been described before and involves mutation in the central loop of domain V of the 23S rRNA (G 2576 T was the most common substitution), with the level of resistance depending on the number of mutated 23S rRNA copies.…”

Section: Discussionsupporting

confidence: 71%

Multistep Resistance Development Studies of Ceftaroline in Gram-Positive and -Negative Bacteria

Clark

McGhee

Appelbaum

et al. 2011

Self Cite

Ceftaroline, the active component of the prodrug ceftaroline fosamil, is a novel broad-spectrum cephalosporin with bactericidal activity against Gram-positive and -negative isolates. This study evaluated the potential for ceftaroline and comparator antibiotics to select for clones of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis with elevated MICs. S. pneumoniae and S. pyogenes isolates in the present study were highly susceptible to ceftaroline (MIC range, 0.004 to 0.25 g/ml). No streptococcal strains yielded ceftaroline clones with increased MICs (defined as an increase in MIC of >4-fold) after 50 daily passages. Ceftaroline MICs for H. influenzae and M. catarrhalis were 0.06 to 2 g/ml for four strains and 8 g/ml for a ␤-lactamase-positive, efflux-positive H. influenzae with a mutation in L22. One H. influenzae clone with an increased ceftaroline MIC (quinolone-resistant, ␤-lactamase-positive) was recovered after 20 days. The ceftaroline MIC for this isolate increased 16-fold, from 0.06 to 1 g/ml. MICs for S. aureus ranged from 0.25 to 1 g/ml. No S. aureus isolates tested with ceftaroline had clones with increased MIC (>4-fold) after 50 passages. Two E. faecalis isolates tested had ceftaroline MICs increased from 1 to 8 g/ml after 38 days and from 4 to 32 g/ml after 41 days, respectively. The parental ceftaroline MIC for the one K. pneumoniae extended-spectrum ␤-lactamase-negative isolate tested was 0.5 g/ml and did not change after 50 daily passages.

“…Vancomycin has been used as a comparator in a significant number of multipassage studies (3,4,6,7,20,21), and the emergence of resistance has been reported in only one of these (3). In our study, the initial MIC for vancomycin increased Ͼ4-fold in two of the four strains tested.…”

Section: Discussionmentioning

confidence: 99%

“…The topical antibiotics mupirocin, fusidic acid, and retapamulin were selected as direct comparators to XF-73, and vancomycin, which has been previously demonstrated to be refractive to the emergence of mutational resistance in multipassage studies (3,6,7,20,21), was also in-cluded. As the mechanism of action (MOA) of daptomycin (16,30) is also proposed to act on the bacterial membrane, it was also tested against USA300, and molecular genetic studies to characterize the mechanism(s) of daptomycin resistance in isolated clones were also undertaken.…”

mentioning

confidence: 99%

Investigation of the Potential for Mutational Resistance to XF-73, Retapamulin, Mupirocin, Fusidic Acid, Daptomycin, and Vancomycin in Methicillin-Resistant Staphylococcus aureus Isolates during a 55-Passage Study

Farrell

Robbins

Rhys‐Williams³

et al. 2011

XF-73 is a dicationic porphyrin drug with rapid Gram-positive antibacterial activity currently undergoing clinical trials for the nasal decolonization of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA). In multistep (55-passage) resistance selection studies in the presence of subinhibitory concentrations of XF-73, retapamulin, mupirocin, fusidic acid, and vancomycin against four Network on Antimicrobial Resistance in Staphylococcus aureus MRSA strains, there was no >4-fold increase in the MIC for XF-73 after 55 passages. In contrast, there was an increase in the MICs for retapamulin (from 0.25 g/ml to 4 to 8 g/ml), for mupirocin (from 0.12 g/ml to 16 to 512 g/ml), for fusidic acid (from 0.12 g/ml to 256 g/ml), and for vancomycin (from 1 g/ml to 8 g/ml in two of the four strains tested). Further investigations using S. aureus NRS384 (USA300) and daptomycin demonstrated a 64-fold increase in the MIC after 55 passages (from 0.5 g/ml to 32 g/ml) with a >4-fold increase in the MIC obtained after only five passages. Sequencing analysis of selected isolates confirmed previously reported point mutations associated with daptomycin resistance. No cross-resistance to XF-73 was observed with the daptomycin-resistant strains, suggesting that whereas the two drugs act on the bacterial cell membrane, their specific site of action differs. XF-73 thus represents the first in a new class of antibacterial drugs, which (unlike the comparator antibiotics) after 55 passages exhibited a <4-fold increase in MIC against the strains tested. Antibacterial drugs with a low propensity for inducing bacterial resistance are much needed for the prevention and treatment of multidrugresistant bacteria both within and outside the hospital setting.