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

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.Despite the introduction of antibiotics such as penicillin in the 1940s and approval of new classes, such as cyclic lipopeptides like daptomycin, infectious disease continues worldwide. Antibiotic resistance is increasing, and reliance on existing scaffolds is not sufficient to combat multidrug resistance (23, 33). Staphylococcus aureus has become a primary concern among antibiotic-resistant infectious disease agents, with methicillin-resistant S. aureus (MRSA) infections claiming 19,000 lives in the United States per year and costing the United States $3 to 4 billion dollars per year (18). As the incidence of MRSA has shifted from hospital settings to the community at large, treatment preferences have moved to oral formulations and have impacted drug resistance profiles. Community-acquired MRSA is showing increasing resistance to fluoroquinolones and clindamycin, and in response clinicians are shifting to doxycycline, linezolid, or trimethoprim-sulfamethoxazole as the best options for outpatient treatment (21, 30).While treatment preferences are adjusting to isolated outbreaks, S. aureus is increasingly resistant to trimethoprim (TMP) (13). TMP targets the enzyme dihydrofolate reductase (DHFR), a critical component of folate metabolism, and is required for continued nucleic acid synthesis (6). DHFR is a good target because of the specificity that can be achieved; however, bacteria have developed resistance mechanisms that include acquisition of plasmid-derived (though catalytically inferior) versions of DHFR, leading to high-level resistance. Alternatively, point mutations of the chromosomal DHFR have been demonstrated to confer intermediate resistance, such as the S. aureus DHFR (saDHFR) residue Phe 98 to Tyr (F98Y) mutation (13). While other mutations are frequently found with F98Y, it alone can increase the MIC of TMP by at least 1 order of magnitude. In efforts to compensate for a lower inhibitory action, TMP is frequently combined with a sulfa inhibitor of the enzyme preceding DHFR, termed dihydropterin synthase (DHPS). Unfortunately, sulfa drugs have notoriously poor pharmacokinetics and can have unpleasant, and sometimes fatal, side effects (15).RAB1 is a lead compound under investigation for treatment of inhalation anthrax. RAB1''s structure is modeled on trimethoprim but is extended by addition of an acryloyl linker and phthalazine moiety, including a propyl group at a stereogenic carbon. This results in approximately 40% more surface area than TMP, which provides critical contact regions for further interaction with the binding site. Specific structural features of RAB1 allow unique contacts with the protein that can overcome natural and induced resistance. For B. anthracis, the half-maximal inhibitory concentration (IC₅₀) of purified DHFR enzyme with TMP is 77 μM, indicating a natural resistance, while with RAB1 it is ≈60 nM (3). Selectivity for the binding site in B. anthracis DHFR is controlled by the large hydrophobic phthalazine moiety, which is embedded within and causes extension of the binding site (4). 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 mutations of chromosomal DHFR to inhibitors in the dihydrophthalazine series (7, 9).We have characterized the broad-spectrum applicability of RAB1 and its particular effectiveness against S. aureus, including MRSA and vancomycin-resistant S. aureus (VRSA) strains. Both enantiomers of RAB1 show potent antimicrobial activities, as indicated by favorable MICs and IC₅₀s. The impact of the TMP resistance-conferring mutation F98Y is negligible on RAB1 activity and preferentially decreases binding of the R-enantiomer. The X-ray crystal structures of RAB1 complexed to S. aureus DHFR reveal two conformations for the large phthalazine group. While one conformation resides in the known binding site, the other sits in a shallow surface cavity that has been previously undocumented as susceptible to inhibitor binding. This allows an increase in binding strength and provides an explanation for the lack of enantiomeric preference. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development, as well as insights into the mechanism of action of phthalazine-based inhibitors.