Antibacterial Antifolates: from Development Through Resistance to the Next Generation
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Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Antibacterial Antifolates: 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 bacteria to synthesize vital building blocks required for proliferation. Therapeutic agents targeting enzymes in this cycle, such as trimethoprim and sulfamethoxazole, 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 dihydrofolate reductase 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 Antibiotics and Antibiotic 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 1 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press A. Estrada et al. dyes. Bayerchemists discovered Prontosil, 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 prodrug and became folates to treat bacterial infections (Darmstadt metabolized to the active agent, sulfanilamide, 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 2 Cite this article as Cold Spring Harb Perspect Med 2016;6:a028324 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press 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 antifolate UCP 1037 Pyridazine DHPS inhibitor Figure 2. Antifolates. Clinically approved antibacterial antifolates: trimethoprim and sulfamethoxazole. Preclinical and experimental antifolates: dihydrofolate reductase (DHFR) inhibitors iclaprim 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