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Antibacterial : From Development through Resistance to the Next Generation

Alexavier Estrada, Dennis L. Wright, and Amy C. Anderson

Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269 Correspondence: [email protected]; [email protected]

The folate cycle is one of the key metabolic pathways used by to synthesize vital building blocks required for proliferation. Therapeutic agents targeting enzymes in this cycle, such as and , are among some of the most important and continually used antibacterials to treat both Gram-positive and Gram-negative pathogens. As with all antibacterial agents, the emergence of resistance threatens the continued clinical use of these life-saving drugs. In this article, we describe and analyze resistance mechanisms that have been clinically observed and review newer generations of preclinical compounds designed to overcome the molecular basis of the resistance.

nhibitors of the folate biosynthetic pathway phosphate (NADPH), as the stoichiometric re- Ihave been successful drugs since the 1940s ducing agent bound in a site immediately adja- when sulfa powder was first applied topically cent to the folate-binding site. These early steps to soldier’s wounds to prevent infection on in the formation of dihydrofolate are selective the battlefield. The folate pathway is essential for bacteria as mammalian cells obtain folic acid in the synthesis of one-carbon donors needed from dietary sources and then sequentially re- for the production of deoxythymidine mono- duce it to tetrahydrofolate using DHFR. There- phosphate (dTMP), purine nucleotides, and fore, antibacterial agents targeting steps before the amino acids methionine and histidine. In DHFR have inherent selectivity, whereas subse- bacteria, dihydrofolate is first synthesized quent targets require the design of species- from early precursor molecules. Most notably, selective agents. The folate cofactors are then

www.perspectivesinmedicine.org the bacterial enzyme, dihydropteroate syn- part of a critical cycle from which the essential thase (DHPS) catalyzes the formation of 7,8- metabolites dTMP, purine nucleotides, histi- dihydropteroate from para-aminobenzoic acid dine, and methionine required for bacterial and 6-hydroxymethyl-7,8,-dihydropterin pyro- growth and division are produced. phosphate (Fig. 1). One additional biosynthetic Blockade of the folate pathway using in- transformation in the bacteria results in the for- hibitors called “antifolates” results in an effec- mation of dihydrofolate, which is reduced by tive “thymine-less death” for the bacterial cell. the essential enzyme The earliest programs to discover antibacterials (DHFR), to tetrahydrofolate. DHFR uses the commenced in the early 1930s and centered on cofactor, nicotinamide adenine dinucleotide antibacterial activity observed with synthetic

Editors: Lynn L. Silver and Karen Bush Additional Perspectives on and Resistance available at www.perspectivesinmedicine.org Copyright # 2016 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a028324 Cite this article as Cold Spring Harb Perspect Med 2016;6:a028324

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dyes. Bayerchemists discovered , one of methoprim and sulfamethoxazole have been the first systemic antibacterial agents to become one of the few truly synergistic antibacterial widely used (van Miert 1994). Later, it was combinations and the only clinically used anti- found that Prontosil is a and became folates to treat bacterial infections (Darmstadt metabolized to the active agent, , 1997; Stevens et al. 2005; Gorwitz et al. 2006; which was identified as a mimic of the para- Nathwani et al. 2008; Frei et al. 2010). aminobenzoic acid substrate and a potent in- In this review, we will describe the common hibitor of DHPS. Many additional inhibitors mechanisms of resistance to both trimethoprim of DHPS, such as sulfamethoxazole (Fig. 2), fol- and sulfamethoxazole, drawing comparisons lowed and were synthesized to improve pharma- between resistant proteins when possible to un- cokinetic parameters, reduce side effects, and derstand the limitations of the enzymes in de- overcome early resistance (Skold 2010). During veloping resistance and to pose possibilities the 1940s, George Hitchings and Gertrude Elion to use that knowledge to overcome resistance. determined that substituted 2,4-diaminopyri- New research to develop DHFR and DHPS in- midines also interfere with folate metabolism. hibitors has focused on overcoming current They synthesized trimethoprim, the first inhib- mechanisms of resistance and providing a next itor of DHFR (Fig. 2). Trimethoprim is very se- generation of antifolates. lective for some bacterial DHFRenzymes such as Staphylococcus aureus and Escherichia coli over human DHFR, a highly potent antibacterial and RESISTANCE TO TRIMETHOPRIM orally bioavailable. One noteworthy feature of During the 1980s, resistance to Bactrim began trimethoprim (TMP) affinity is its significant to occur in both the hospital and community cooperativity in binding with the NADPH co- settings (Then et al. 1992; Dale et al. 1997). Two factor. Soon after, Bactrim, a combination of prevalent modes of resistance arose: chromo- trimethoprim and sulfamethoxazole, became a somal mutations in the dfrB and folP genes of widely used therapy to treat infections caused S. aureus, which encode for DHFR and DHPS, by both Gram-positive (skin and soft-tissue in- respectively, and the horizontal transfer of plas- fections and pneumonia) and Gram-negative mid-encoded, trimethoprim-resistant dfr genes (urinary tract infections) bacteria. To date, tri- or sulfamethoxazole-resistant sul genes in both

Dihydropteroate pyrophosphate Glutamate + + para-aminobenzoic

www.perspectivesinmedicine.org Dihydropteroate Dihydrofolate acid Dihydrofolate Trimethoprim Dihydrofolate reductase Dihydropteroate synthase synthase Tetrahydrofolate

Serine Sulfamethoxazole dTMP hydroxymethyltransferase

Thymidylate 5,10-methylene synthase tetrahydrofolate

dUMP Methionine (SAM) Histidine purines Figure 1. The folate cycle in bacteria. Enzymes are shown in green, key products in blue, and inhibitors in red. Dihydropteroate synthase (DHPS), a critical enzyme in the formation of dihydrofolate, is inhibited by sulfa- methoxazole, and dihydrofolate reductase (DHFR) is inhibited by trimethoprim. dTMP, Deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; SAM, S-adenosylmethionine

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Antibacterial Antifolates

NH 2 N O OMe O O N S N H H N N OMe 2 H N OMe 2 Trimethoprim Sulfamethoxazole

O O NH2 O O NH2 O N HN COOH N H N N N 2 H2N N N H2N N OMe H OMe N

Iclaprim Propargyl-linked UCP 1037 Pyridazine DHPS inhibitor

Figure 2. Antifolates. Clinically approved antibacterial antifolates: trimethoprim and sulfamethoxazole. Preclinical and experimental antifolates: dihydrofolate reductase (DHFR) inhibitors and propargyl- linked antifolates, and dihydropteroate synthase pyridazine (DHPS) inhibitors.

Gram-positive and Gram-negative bacteria dfrA and called S1 DHFR was isolated and (Amyes 1989; Amyes et al. 1992; Skold 2010). shown to confer resistance to strains in Australia A discussion of the chromosomal mutations and the United States (Amyes 1989). S1 DHFR, and plasmid-encoded resistant proteins follows; which may originate from Staphylococcus epi- comparisons of these resistant proteins (Fig. 3) dermis, possesses three key residue substitu- may shed light on common mechanisms to de- tions, F98Y, V31I, and G43A, that have been crease drug affinity. shown to be responsible for the majority of In 1997, Dale et al. (1997) reported that TMP resistance (Fig. 3) (Dale et al. 1995). It is chromosomal mutations in the dfrB gene en- noteworthy that this plasmid-encoded DHFR coding DHFR in S. aureus were responsible recapitulates the key F98Y mutation observed for minimum inhibitory concentration (MIC) in the chromosome. A detailed kinetic and changes of 256-fold. Of the chromosomal structural comparative study of the wt and S1 mutations, the active-site mutation, F98Y, enzymes revealed that the S1 enzyme indepen- www.perspectivesinmedicine.org posed the greatest problem, conferring .400- dently reduces the interactions of both TMP fold resistance to trimethoprim (IC50 values for and NADPH such that the binding synergy be- TMP against the wild-type (wt) and F98Y en- tween the two is significantly reduced by 750- zymes are 0.01 and 4.1 mM, respectively). The fold. A crystal structure of S1 with TMP revealed F98Y mutation most frequently occurs with an six molecules in the asymmetric unit, only three additional compensatory mutation, either of which were the ternary structure. The other H30N or H149R, that bring the fitness of the three S1 proteins bound only TMP, indicating enzyme to near wild-type levels and increase the the altered interactions of the S1 enzyme with degree of resistance to trimethoprim by 800- to NADPH (Heaslet et al. 2009). 2400-fold over wt. At the time the study was In 2005, a second exogenous DHFR encod- published, 88% of resistant strains possessed ed by dfrG and called S3 DHFR, was discovered MIC changes of 256-fold, suggesting the in a collection of 43 clinical isolates of TMP- widespread presence of chromosomal muta- resistant strains of methicillin-resistant Staph- tions in the dfrB gene. ylococcus aureus (MRSA) from Thailand and In parallel, a transposon-encoded trimeth- 244 isolates from Japan (243 of these were oprim-resistant enzyme encoded by the gene TMP-sensitive; one was resistant) (Sekiguchi

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A. Estrada et al.

28 31 SaWT -----MTLSILVAHDLQRVIGFENQLPWHLPNDLKHVKKLSTGHTLVMGRKTFESIGKPL 55 DfrA (S1) -----MTLSIIVAHDKQRVIGYQNQLPWHLPNDLKHIKQLTTGNTLVMARKTFNSIGKPL 55 DfrG (S3) -----MKVSLIAAMDKNRVIGKENDIPWRIPKDWEYVKNTTKGHPIILGRKNLESIGRAL 55 KpWT ------MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVVMGRLTWESIGRPL 54 DfrA1 -----MKISLMVAISKNGVIGNGPDIPWSAKGEQLLFKAITYNQWLLVGRKTFESMGA-L 54 DfrA12 MNSESVRIYLVAAMGANRVIGNGPNIPWKIPGEQKIFRRLTEGKVVVMGRKTFESIGKPL 60 DfrA17 -----MKISLISAVSENGVIGSGPDIPWSVKGEQLLFKALTYNQWLLVGRKTFDSMGV-L 54 92 98 SaWT PNRRNVVLTSDT-SFNVEGVDVIHSIEDIYQLP----GHVFIFGGQTLFEEMIDKVDDMY 110 DfrA (S1) PNRRNVVLTNQA-SFHHEGVDVINSLDEIKELS----GHVFIFGGQTLYEAMIDQVDDMY 110 DfrG (S3) PDRRNIILTRDK-GFTFNGCEIVHSIEDVFELCKNE-EEIFIFGGEQIYNLFFPYVEKMY 113 KpWT PGRKNIVISSKP-GS-DDRVQWVSSVEEAIAACGDV-EEIMVIGGGRVYEQFLPKAQKLY 111 DfrA1 PNRKYAVVTRSSFTSDNENVLIFPSIKDALTNLKKITDHVIVSGGGEIYKSLIDQVDTLH 114 DfrA12 PNRHTLVISRQA-NYRATGCVVVSTLSHAIALASELGNELYVAGGAEIYTLALPHAHGVF 119 DfrA17 PNRKYAVVSKNGISSSNENVLVFPSIENALKELSKVTDHVYVSGGGQIYNSLIEKADIIH 114

SaWT ITVIEGK-FRGDTFFPPYTFEDWEVASSVEGKLDEKNTIPHTFLHLIRKK------159 DfrA (S1) ITVIDGK-FQGDTFFPPYTFENWEVESSVEGQLDEKNTIPHTFLHLVRRKGK------161 DfrG (S3) ITKIHHE-FEGDTFFPEVNYEEWNEVFAQKGIKNDKNPYNYY-FHVYERKNLLS---- 165 KpWT LTHIDAE-VEGDTHFPDYDPDEWESVFSEFHDADAQNSHSYCFEILERR------159 DfrA1 ISTIDIE-PEGDVYFPEI-PSNFRPVFTQDFASNI----NYSYQ-IWQKG------157 DfrA12 LSEVHQT-FEGDAFFPMLNETEFELVSTETIQAVI----PYTH-SVYARRNG------165 DfrA17 LSTVHVE-VEGDIKFPIM-PENFNLVFEQFFMSNI----NYTYQ-IWKKG------157

Figure 3. Sequence alignment for trimethoprim-sensitive (SaWT and KpWT) and trimethoprim-resistant dihydrofolate reductases (DHFRs). S1 DHFR and S3 DHFR are found in Gram-positive bacteria. DfrA1, DfrA12, and DfrA17 are found in Gram-negative bacteria. Residues colored in red are located in the inhibi- tor-binding pocket. As Klebsiella pneumoniae and Escherichia coli sequences are almost identical, only one sequence is shown here.

et al. 2005). TMP-resistant isolates possessing bacteria, surveillance of resistance in this popu- dfrG showed greatly elevated MIC values of at lation is also actively monitored. In Gram-neg- least 512 mg/mL. The dfrG gene, while original- ative bacteria, especially the Enterobacteriaceae ly horizontally transferred via plasmid, incor- E. coli and Klebsiella pneumoniae, a different porated into the genome. Isolation and char- group of trimethoprim-resistant extrachromo- acterization of the S3 enzyme showed that, somal DHFR proteins are horizontally trans- www.perspectivesinmedicine.org although KM values for DHF and NADPH ferred between bacteria. In Gram-negative bac- were similar to the wt enzyme, TMP affinity teria, plasmid-encoded TMP resistance was first was weakened by 20,000-fold (IC50 values of reported in 1992 (Amyes et al. 1992) with the the wt and S3 enzyme were 0.013 and 254 mM, description of two types of plasmid-encoded respectively). MIC values for TMP using E. coli DHFR enzymes. Each type was highly resistant transformants with the new plasmid were sig- to trimethoprim, conferring MIC values nificantly increased, validating that the presence .1mM, but differed in their protein expression of dfrG confers TMP resistance. All isolates from levels, with type 1 enzymes producing 10-fold Thailand were resistant to TMP and possessed greater levels of protein than the chromosom- dfrG; only one isolate from Japan was resistant al enzyme. Two studies, one conducted in to TMP and contained dfrG (Sekiguchi et al. Sweden (Brolund et al. 2010) and the other in 2005). In a second publication, expansion of Western Europe and Canada (Blahna et al. dfrG to Africa and transfer to European travelers 2006), surveyed the prevalence of type 1 or from Africa was described (Nurjadi et al. 2014). type 2 integron-encoded trimethoprim-resis- As Bactrim is first-line treatment for com- tant dfr genes. The Swedish study concurred mon infections caused by Gram-negative that the type 1 gene dfrA1 is most prevalent

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Antibacterial Antifolates

and occurred in 34% of resistant isolates. Of the hydrophobic interactions that support the di- 350 isolates studied in the European/Canadian aminopyrimidine ring and linker (Fig. 4A). study, 66% possessed an integron-associated Interestingly, this residue is an isoleucine in S1 dfr allele. The gene dfrA1 was again the most DHFR, which is known to contribute to TMP prevalent (40%), followed by a second gene, resistance (Heaslet et al. 2009). In the Gram- dfrA17 (31%). negative enzymes, Phe at position 31 is balanced Interestingly, although the sequences of the by less bulky residues on the opposite side of the trimethoprim-resistant DHFR proteins diverge, active site (Fig. 4B): Ile in the wt, Ser in DfrA1, the proteins share several motifs that distinguish Ala in DfrA12, and Ser in DfrA17. These smaller the TMP-resistant proteins from the TMP-sen- residues (serine and alanine) in the DfrA1, sitive (Fig. 3). For example, position 28 is a DfrA12, and DfrA17 are likely to decrease affin- leucine in S. aureus (wt), E. coli, and K. pneumo- ity with the antifolates relative to the isoleucine niae (wt) but changes to a tryptophan in S3 in the wild-type. DHFR and is consistently a glutamine in all of Interestingly, position 98 is a phenylalanine the TMP-resistant Gram-negative Dfr proteins. in S. aureus (wt) and corresponds to a tyrosine This switch from a leucine to a bulky trypto- in S1 and S3 DHFR that are responsible for phan or polar glutamine is likely to affect posi- TMP-resistance in Gram-positive bacteria tioning of the diaminopyrimidine ring as well (Fig. 4A,B). From the early clinical observa- as the hydrophobic trimethoxyphenyl ring (Fig. tions, the chromosomal F98Y mutation is also 4A,B). These effects were in fact observed in the known to cause significant TMP resistance in structure of DfrA1 bound to an experimental S. aureus DHFR. More detailed studies have antifolate (Lombardo et al. 2015). shown that this residue change greatly affects Position 31 is valine in S. aureus (wt) but a the position and affinity of the cofactor, phenylalanine in K. pneumoniae (wt) or any of NADPH (Heaslet et al. 2009) as well as the ki- the Gram-negative DfrA proteins. The valine in netics and entropy of binding. Recent structural S. aureus and S3 is balanced by Phe 92, which is and biochemical studies investigating the wild- on the opposite side of the active site, forming type and F98Y enzymes show that the enzyme

AB

F/Y98 Y100 NADPH NADPH www.perspectivesinmedicine.org

TMP D/E27

F92 V/I31 F31

L/Q28 L28

Figure 4. Structural comparisons of wild-type and trimethoprim-resistant DHFR. Structural superposition of (A) Staphylococcus aureus (wild-type [wt]) (pink) bound to nicotinamide adenine dinucleotide phosphate (NADPH) (yellow) and trimethoprim (TMP) (blue) with S1 dihydrofolate reductase (DHFR) (green) bound to NADPH and TMP (not shown) (Heaslet et al. 2009), and (B) Klebsiella pneumoniae (wt) (dark blue) bound to NADPH (orange) and a propargyl-linked antifolate (PLA) (not shown) with DfrA1 (cyan) bound to NADPH and a PLA (green) (Lombardo et al. 2015).

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A. Estrada et al.

displays plasticity in the cofactor binding site, SMX is first-line therapy, showed that 24 out of which is enhanced with the F98Y mutation. 106 isolates were resistant to TMP-SMX. The This plasticity affects a distribution of ternary sul1 or sul2 gene was present in 12 or 14 isolates, states that includes an alternate bound config- respectively (Kaur et al. 2015). uration of the cofactor (Keshipeddy et al. 2015). Resistance to antibacterial agents can take In contrast, both the wild-type E. coli and K. many forms including efflux, drug-modifying pneumoniae DHFR enzymes that are sensitive enzymes, changes in cell membrane permeabil- to TMP as well as the plasmid-encoded TMP- ity, and alterations of the drug target. It appears resistant DfrA1, DfrA12, and DfrA17 all natively that the majority of resistance to currently used possess tyrosine at position 98, suggesting that antifolates is heavily dependent on alterations of compensatory and subtle differences exist be- the drug target and the introduction of mobile tween the Gram-positive and the Gram-nega- elements that carry a resistant copy of the target tive DHFR enzymes. as opposed to mechanisms that alter drug con- centration. This knowledge of the mechanisms of resistance coupled with detailed structural RESISTANCE TO SULFAMETHOXAZOLE and biochemical analysis underlies the design Resistance to sulfamethoxazole also arose early of next-generation antifolates. after introduction and began to be monitored. As with trimethoprim, resistance to sulfame- COMPOUNDS IN DEVELOPMENT thoxazole arises through combinations of chro- mosomal mutations and plasmid-encoded Fortunately, there continues to be significant copies of resistant genes. Several point mutations development of new antifolates intended to in the folP gene encoding DHPS in S. aureus, overcome existing resistance mechanisms. De- have been reported in clinical isolates. Seven of velopment has focused on new DHFR inhibi- the nine resistant strains analyzed showed at least tors including iclaprim and the propargyl- 13 residues that differ from wild-type; the re- linked antifolates as well as new DHPS inhibi- maining two isolates have two residue differ- tors intended to bind and inhibit the pterin ences. Crystal structures of S. aureus (Hampele pocket of the enzyme, thus avoiding resistance et al. 1997), Yersinia pestis (Yun et al. 2012), or mutations already present. Mycobacterium tuberculosis DHPS (Baca et al. Iclaprim (Fig. 2), originally developed by 2000) show that most of the mutations impli- Roche and licensed by Arpida for clinical ad- cated in resistance are spread across the surface vancement (Hawser et al. 2006), was designed of the protein or in flexible loops. Resistance to overcome the resistance conferred by point www.perspectivesinmedicine.org mutations F28L/I and P64S (E. coli numbering) mutations in the chromosomal gene and to be are found in loop 1 and loop 2, respectively, effective against other TMP-resistant organ- close to the active site and affect the para- isms. The compound showed potent activity aminobenzoic acid (PABA) binding site. Yun against MRSA, TMP-resistant MRSA, Strepto- et al. (2012) further notes that mutations at coccus pyogenes (MIC values between 0.03 mg/ positions F33 and P69 are likely to affect inhib- mL and 0.06 mg/mL), as well as Gram-negative itor but not substrate binding, which may make bacteria such as Enterococcus and Haemophilus these sites more susceptible to resistance-con- influenza (MIC values 0.12 and 0.5 mg/mL, re- ferring mutations. spectively) (Schneider et al. 2003). Further- In addition to chromosomal gene muta- more, the propensity of iclaprim to induce the tions, sulfamethoxazole resistance can be trans- formation of resistance mutations is reported to ferred by integrons carrying the sul1, sul2,or be very low. Iclaprim advanced through phase I, sul3 genes (Skold 1976; Blahna et al. 2006) en- II, and III clinical trials focused on complicated coding insensitive forms of DHPS. In fact, a skin and soft tissue infections. Unfortunately, collection of 106 clinical isolates of Stenotropho- some cardiotoxicity issues presented during monas maltophilia from India, for which TMP- the trials (Sincak and Schmidt 2009) and

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Antibacterial Antifolates

more importantly, the drug did not meet the a crystal structure of Bacillus anthracis DHPS regulatory metrics of non-inferiority relative bound to a lead pterin (Hevener et al. 2012), to in place at the time. Recently, a bio- Lee and coworkers synthesized several pyrida- technology company, Motif Bio (see motifbio zine derivatives intended to optimize different .com/iclaprim), received FDA approval to re- functional groups in the lead (Fig. 2). Using a open the phase III clinical trials for acute bacte- combination of enzyme assays, ITC, SPR, and rial skin and skin structure infections as well as crystallography, they show that a demethylated hospital-acquired bacterial pneumonia. pyridazine core is a pterin mimic for DHPS A series of compounds known as the prop- (Zhao et al. 2012). In parallel work, they show argyl-linked antifolates (PLAs; example shown that a compound targeted to the DHPS dimer in Fig. 2) is currently under development to interface acts as an allosteric inhibitor of the inhibit both Gram-positive and Gram-negative enzyme, opening a new area of design and in- bacteria; they are specifically designed to target tervention (Hammoudeh et al. 2014). both trimethoprim-sensitive and trimetho- prim-resistant enzymes. Using a structure- based approach founded on the determination CONCLUSION of several tens of structures of DHFR from many Despite the fact that trimethoprim and sulfame- species, including S. aureus (Frey et al. 2009), thoxazole have been in continued use for over mutant forms of S. aureus (Frey et al. 2009, 60 years, these classes of antibacterials have 2010; Keshipeddyet al. 2015), E. coli, K. pneumo- not undergone the sustained medicinal chemis- niae (Lamb et al. 2014), and plasmid-encoded try efforts that have led to multiple generations trimethoprim-resistant DHFR such as DfrA1 of other important classes such as b-lactams (Lombardo et al. 2015), new DHFR inhibitors or fluoroquinolones. Although resistance to were designed to overcome mutations that cause these agents has appeared, they remain some TMP resistance as well as whole enzymes that are of the most important first-line therapeutics horizontally transferred for resistance. The de- for common Gram-positive and Gram-negative velopment of these compounds has led to very pathogens. However, increased use coupled with potent inhibitors of wild-type and mutant globalization demands the development of new forms of S. aureus DHFR as well as the growth antifolates that will continue to allow this im- of the S. aureus (wild-type and mutant) bacteria. portant class to be clinically effective. Some of the most potent compounds have MIC values of 0.0391 mg/mL against the wild-type and 0.625 mg/mL against S. aureus with the ACKNOWLEDGMENTS www.perspectivesinmedicine.org F98Y mutation (Keshipeddy et al. 2015). In addition, many of the compounds show activ- The authors acknowledge support from the ity against Gram-negative bacteria and show po- National Institutes of Health (NIH) (AI104841 tency against the trimethoprim-resistant DfrA1 and AI111957). (Lombardo et al. 2015). Early investigations into the propensity to incur resistance mutations show that the compounds generally have very REFERENCES 29 low resistance frequencies between 1.2 Â 10 Amyes S. 1989. The success of plasmid-encoded resistance and 8.9 Â 10210 (Frey et al. 2012). genes in clinical bacteria. J Med Microbiol 28: 73–83. There has also been considerable effort to Amyes S, Towner K, Young H. 1992. Classification of plas- mid-encoded dihydrofolate reductases conferring tri- develop new inhibitors of DHPS. Switching at- methoprim resistance. J Med Microbiol 36: 1–3. tention from the para-aminobenzoic acid site Baca A, Sirawaraporn R, Turley S, Sirawaraporn W, Ho W. where the sulfa compounds bind, medicinal 2000. Crystal structure of Mycobacterium tuberculosis 6- chemistry work has focused on the pocket that hydroxymethyl-7,8-dihydropteroate synthase in complex with pterin monophosphate: New insight into the enzy- binds the partner substrate, dihydropterin py- matic mechanism and sulfa-drug action. J Mol Biol 302: rophosphate (DHPP) (Zhao et al. 2012). Using 1193–1212.

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Antibacterial Antifolates

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Antibacterial Antifolates: From Development through Resistance to the Next Generation

Alexavier Estrada, Dennis L. Wright and Amy C. Anderson

Cold Spring Harb Perspect Med 2016; doi: 10.1101/cshperspect.a028324 originally published online June 28, 2016

Subject Collection Antibiotics and Antibiotic Resistance

Fosfomycin: Mechanism and Resistance The Whys and Wherefores of Antibiotic Lynn L. Silver Resistance Cameron R. Strachan and Julian Davies Pleuromutilins: Potent Drugs for Resistant Bugs β-Lactamases: A Focus on Current Challenges −−Mode of Action and Resistance Robert A. Bonomo Susanne Paukner and Rosemarie Riedl Appropriate Targets for Antibacterial Drugs Approved Glycopeptide Antibacterial Drugs: Lynn L. Silver Mechanism of Action and Resistance Daina Zeng, Dmitri Debabov, Theresa L. Hartsell, et al. Lincosamides, Streptogramins, Phenicols, and Mechanism of Action and Resistance to Pleuromutilins: Mode of Action and Mechanisms Daptomycin in Staphylococcus aureus and of Resistance Enterococci Stefan Schwarz, Jianzhong Shen, Kristina Kadlec, William R. Miller, Arnold S. and Cesar A. et al. Arias Resistance to Macrolide Antibiotics in Public Polymyxin: Alternative Mechanisms of Action and Health Pathogens Resistance Corey Fyfe, Trudy H. Grossman, Kathy Kerstein, et Michael J. Trimble, Patrik Mlynárcik, Milan Kolár, et al. al. Bacterial Protein Synthesis as a Target for Topoisomerase Inhibitors: Fluoroquinolone Antibiotic Inhibition Mechanisms of Action and Resistance Stefan Arenz and Daniel N. Wilson David C. Hooper and George A. Jacoby Antibacterial Antifolates: From Development β-Lactams and β-Lactamase Inhibitors: An through Resistance to the Next Generation Overview Alexavier Estrada, Dennis L. Wright and Amy C. Karen Bush and Patricia A. Bradford Anderson Antibacterial Drug Discovery Targeting the , Alone and in Combination Lipopolysaccharide Biosynthetic Enzyme LpxC David M. Rothstein Alice L. Erwin

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