bioRxiv preprint doi: https://doi.org/10.1101/504357; this version posted December 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Structural and Biochemical Studies of Dihydrofolate Reductase from Streptococcus pyogenes as a Target for Antifolate Antibiotics Behnoush Hajian1, Jolanta Krucinska1, Michael Martins2, Narendran G-Dayanan1, Kishore Viswanathan1, Sara Tavakoli2, Dennis Wright1 1Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269 2Department of Cellular and Molecular Biology, University of Connecticut, Storrs, CT 06269 Corresponding author: Dennis Wright, [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/504357; this version posted December 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. ABSTRACT Streptococcus pyogenes, a beta-hemolytic bacterium, causes a wide spectrum of infections in human including pharyngitis, tonsillitis, scarlet fever, rheumatic fever, and necrotizing fasciitis. Streptococcal infections can also exist as co-infection with methicillin resistant Staphylococcus aureus (MRSA). Trimethoprim-sulfamethoxazole (TMP-SMX) combination has been used for treatment of S. pyogenes and MRSA co-infection. However, resistance to TMP, an inhibitor of dihydrofolate reductase enzyme (DHFR), has challenged the efficacy of TMP-SMX combination. We explored the activity of a series of novel DHFR inhibitors against S. pyogenes. This study identified potent inhibitors of DHFR enzyme from S. pyogenes with excellent inhibitory activity against the growth of the live bacteria. We determined, for the first time, the crystal structure of S. pyogenes DHFR which provides structural insights into design and development of antifolate agents against this global pathogen. Key words: Streptococcus pyogenes, folate pathway, dihydrofolate reductase, trimethoprim, MRSA, antifolates 2 bioRxiv preprint doi: https://doi.org/10.1101/504357; this version posted December 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. INTRODUCTION Streptococcus pyogenes, or Lancefield’s lactamase producing bacteria such as group A Streptococcus (GAS), is a Gram- Staphylococcus aureus.4 positive bacterium that causes a diverse Trimethoprim-sulfamethoxazole spectrum of human infections. combination (SXT), one of the most Streptococcal infections are usually mild widely used and cheapest antibacterials such as pharyngitis (strep throat) and in the world, is currently suggested as a impetigo. But if the infection reaches valuable option for treatment of skin and deeper tissues, it can cause invasive soft tissue coinfections with S. pyogenes infections such as necrotizing fasciitis and methicillin-resistant S. aureus (flesh eating disease) and streptococcal (MRSA), when penicillin treatment fails.12 toxic shock syndrome. Superficial GAS However, the emergence of infections can be followed by abnormal trimethoprim (TMP) resistance in S. immune responses which may result in pyogenes isolates have challenged the post-streptococcal sequelae including efficacy of SXT.13,14 TMP is an inhibitor of acute rheumatic fever and acute post- dihydrofolate reductase (DHFR), one of streptococcal glomerulonephritis.1 The the key enzymes in the folate prevalence of severe GAS infections is biosynthetic pathway. The folate 18.1 million cases, with 1.78 new cases pathway is essential in the synthesis of and 517 000 deaths each year. The past reduced folates, the one-carbon donors decade has witnessed a global required for the production of resurgence of streptococcal diseases deoxythymidine monophosphate such as skin and soft tissue infections and (dTMP), purine nucleotides, methionine scarlet fever.2,3 and histidine (Figure 1). DHFR catalyzes Although, S. pyogenes in general the reduction of DHF to THF using remains susceptible to most classes of NADPH as an electron donor. Due to its antibiotics, treatment of streptococcal pivotal role in regulating cellular levels of infections is challenged by the rising tide THF and its derivatives, DHFR has served of antimicrobial resistance (AMR).4–7 as an attractive target for many Over the past two decades, there has anticancer, antibacterial, and been an increasing rate of macrolide antiprotozoal drugs.15,16 resistance among S. pyogenes isolates in Herein, we report the activity of a Europe and worldwide.8–11 There has series of propargyl-linked antifolates been also a growing rate of penicillin (PLAs) against S. pyogenes and DHFR failure mostly due to lack of penicillin enzyme from this pathogen (SpDHFR). permeation into the infected tissues and Previously, we have shown that PLAs are co-infection of S. pyogenes with beta- inhibitors of TMP-susceptible and TMP- resistant MRSA isolates.17–20 Screening of 3 bioRxiv preprint doi: https://doi.org/10.1101/504357; this version posted December 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. PLAs against S. pyogenes identified a of the lead PLAs which provides group of novel PLAs with pronounced structural insights into the design of antibacterial activity. Further exploration potent and selective inhibitors. Our data of structure activity relationship (SAR) of strongly support the effort to explore this group has led to the identification of PLAs as promising candidate for design potent inhibitors of SpDHFR enzyme. of novel antifolates against TMP- Here we report for the first-time high resistant S. pyogenes and MRSA resolution crystal structure of SpDHFR in coinfections. a ternary complex with NADPH and one Figure 1. Bacterial folate cycle and related pathways. ADC: aminodeoxy chorismate, PABA: para-aminobenzoic acid, DHPPP: 7,8-dihydropterin pyrophosphate, HMDHP: 6-hydroxymethyl-7,8-dihydropterin, DHNP: 7,8-dihydroneopterine, DHNP-PPPi: 7,8-dihydroneopterine triphosphate, DHP: 7,8-dihydropteroate, DHF: 7,8-dihydrofolate, THF: 5,6,7,8-tetrahydrofolate, DHFR: dihydrofolate reductase, SHMT: serine hydroxymethyltransferase, mTHF: N5,N10-methyleneTHF, 5-mTHF: N5-methylTHF, TS: thymidylate synthase, MS: methionine synthase, SAM: S-adenosyl methionine, SAH: S-adenosyl homocysteine RESULTS method, a series of PLAs were tested for Antimicrobial activity of PLAs against their inhibitory activity against the S. pyogenes. Using broth micro dilution growth of S. pyogenes ATCC 19615 4 bioRxiv preprint doi: https://doi.org/10.1101/504357; this version posted December 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. (Rosenbach), a quality control strain used in a variety of susceptibility assays. PLASs scaffold includes a 2,4- decrease in the potency of the diaminopyrimidine ring (ring A) linked to compounds. As seen with compounds 1 an aryl or heteroaryl system (ring B and and 7, the absence of propargylic methyl C) through a propargyl bridge. Based on leads to improved inhibitory activity the variations in the B- and C-rings, the against SpDHFR (IC50 values of 73 and 57 tested PLAs were categorized into six nM, respectively). In addition, moving general groups (Table 1 and Table S1). the carboxylate from the para (in With only one exception (compound 34), compound 7) to the ortho position (in all the tested compounds exhibited very compound 6) compromised the promising antimicrobial activity against inhibitory effect by almost four-folds (IC50 S. pyogenes with MIC values below 1 values of 57 and 200 nM, respectively). µg/ml. Furthermore, the stereoisomers of S configuration (compounds 2 and 4) Inhibition of S. pyogenes DHFR exhibit similar activity with R isomers enzyme by PLAs. To test whether the (compounds 3 and 5). The lack of inhibitory activity of PLAs against S. apparent stereospecificity is pyogenes cells is mediated through the continuously evident across all the other inhibition of DHFR enzyme, we tested PLAs. evaluated the inhibitory effect of the Compounds in group two feature a compounds against the purified enzyme. pyrimidine C-ring and a chlorine The half maximal inhibitory substitution at 2’ position on the B-ring. concentrations (IC50 values) are shown in Here, compound 8 is the most potent Table 1. Although the potency of the congener with IC50 value of 85 nM. compounds varies, in general there is a Introduction of two methyl groups on the correlation between the enzyme and cell C-ring (compound 10) leads to a modest growth inhibition. SAR analysis of each decrease in inhibitory affinity (IC50 value group of the tested compounds of 193 nM). elucidated key structural features that Based on the same concept, group affect the potency of PLAs against three represents fluorine atoms SpDHFR. substitution on the B-ring. IC50 values for Group one is characterized by PLA- this group range from 40 to 350 nM. Not COOH compounds which feature a surprisingly, the presence of propargylic carboxylic acid substitution on the C- methyl and/or methyl substitutions on ring. The IC50 values observed in this the C-ring reflects the same trend as category varies between 50 to 250 nM. seen with chlorine atom derivatives. Within this group, the presence