Discovery and Characterization of a Class of Pyrazole Inhibitors of

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Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase Nestor O. Concha, Jianzhong Huang, Xiaopeng Bai, Andrew B Benowitz, Pat G. Brady, LaShadric C. Grady, Luz Helena Kryn, David Holmes, Karen Ingraham, Qi Jin, Laura Pothier Kaushansky, Lynn McCloskey, Jeffrey A Messer, Heather O’Keefe, Amish Patel, Alexander L. Satz, Robert H. Sinnamon, Jessica L Schneck, Steven R. Skinner, Jennifer Summerfield, Amy N. Taylor, J. David Taylor, Ghotas Evindar, and Robert A. Stavenger J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00746 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 9, 2016

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Journal of Medicinal Chemistry

Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase Nestor Concha,† Jianzhong Huang,† Xiaopeng Bai,‡ Andrew Benowitz,† Pat Brady,† LaShadric C. Grady,‡ Luz Helena Kryn,ǁ David Holmes,† Karen Ingraham,† Qi Jin,† Laura Pothier Kaushansky,‡ Lynn McCloskey,† Jeffrey A. Messer,‡ Heather O’Keefe,‡ Amish Patel,‡ Alexander L, Satz,‡ Robert H. Sinnamon,† Jessica Schneck,† Steve R. Skinner,‡ Jennifer Summerfield,‡ Amy Taylor,† J. David Taylor,† Ghotas Evindar,*,‡ Robert A. Stavenger*† †

‡

GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, Pennsylvania 19426; GlaxoSmithKline, 830 Winter Street, ǁ Waltham, MA 02451; GlaxoSmithKline, 5 Moore Dr., Research Triangle Park, NC, 27009. KEYWORDS: undecaprenyl pyrophosphate synthase | drug discovery | x-ray crystallography | anti-microbial ABSTRACT: Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through co-crystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.

INTRODUCTION Undecaprenyl pyrophosphate synthase (UppS, EC 2.5.1.31) is a cis-prenyltransferase that catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to form C55-undecaprenyl pyrophosphate (UPP)1. Subsequent removal of the terminal UPP phosphate by C55isoprenol pyrophosphate phosphatase produces undecaprenyl phosphate (UP)2. UppS has been demonstrated to be essential for cell viability in Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae35 due to its role in the synthesis of UP which transports building blocks such as N-acetyl-glucosamine-N-acetylmuramate-pentapeptide across the cell membrane, to allow the synthesis and assembly of peptidoglycans, teichoic acids, and lipopolysaccharides6. A number of crystal structures of the UppS protein have been reported, and the multiple protein conformations (native, substrate-, and product-bound) in these studies highlight the protein’s flexible nature7-12. An important structural observation is a deep and elongated active site that appears to be amenable to small molecule inhibition7,8,11. The essentiality of UppS in many pathogenic bacteria3-5, coupled with an apparently druggable active site, makes it an attractive target for the discovery of new antibacterials. Several groups have sought to identify UppS-inhibitors through techniques including rational design, virtual, and high-throughput screening with variable levels of success9-18. Many of these molecules have hydrophobic moieties that bind in the hydrophobic tunnel with some contain polar groups interacting with the

pyrophosphate-binding site. Although a ‘hot spot’ for binding, we found this pocket was not very conducive to small molecule optimization, vide infra. Encoded Library Technology (ELT) is a robust hit identification platform19 that utilizes DNA-encoded small molecule libraries containing billions of individual potential ligands for affinity-based selections against protein targets.20-22 ELT was used to screen Staphylococcus aureus (S. aureus) UppS, and identified a series of 1,4,5substituted pyrazoles as UppS inhibitors which demonstrated modest whole-cell antimicrobial activity. Biochemical and thermal shift assays confirmed that these compounds bound to recombinant target protein and inhibited the production of UPP in vitro. Bacterial strains with regulated uppS expression showed that the antimicrobial activity was indeed the result of UppS inhibition. Inhibitor co-crystal structures with S. pneumoniae UppS revealed the compounds bound in the large and hydrophobic region at the base of the FPP pocket. Furthermore, we were able to generate resistant mutants of S. aureus grown in the presence of UppS inhibitors, and these mutations mapped to the FPP binding site, offering further confirmation that the antibacterial action is due to inhibition of the UppS. Although the corresponding optimization program did not progress, we have established clear on-target mechanism of action evidence for this class of UppS inhibitors. RESULTS AND DISCUSSION Inhibitor Discovery via ELT Screening. The UppS affinity selections against a collection of ELT libraries were performed using purified, recombinant S. aureus

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UppS. The target protein construct contained N-terminal FLAG and C-terminal Streptavidin Binding Protein (SBP) dual purification tags. Selections were performed by immobilizing the protein via SBP on streptavidin-derivatized Phynexus affinity columns. Three rounds of selection were carried out in parallel with a negative control selection performed using the same protocol, but with no target protein bound to the tips (NTC). Following selection, the output was quantified and the DNA tags amplified and sequenced in order to deconvolute the identity of the S. aureus UppS protein binding templates. The ELT selection data were analyzed in a SpotfireTM cube view (Figure 1a). One series of the hits came from an unbiased 3 cycle diversity library with a theoretical library size of 1.6 million compounds. The cube displayed in Figure 1a shows the family of compounds that have been enriched with two or more independent DNA sequences. The cube axes each represent one building block (BB) used in the synthesis (e.g. BB1, BB2, and BB3, see Figure 1b) in cycles 1, 2 and 3, respectively of the original ELT library synthesis. Thus a pair of BBs which generally leads to target binding will be represented by a line Figure 1a. Each data point is sized proportionally to its copy number during the selections and the display is set to a minimum of 10 unique copies per warhead, indicating significantly enriched binders under the selection conditions used in this study. Although several patterns are evident in Figure 1a, the feature (chemical series) highlighted in the blue oval stood out during the analysis and corresponds to the scaffold described in Figure 1b. The compounds in this feature share a common BB1 (4isopropoxyphenyl-methanamine) and BB2 (1-benzyl-5iodo-1H-pyrazole-4-carboxylic acid) with variability in BB3 which is visualized as a line in the cube in Figure 1a (see Table S1 for additional BB3 SAR obtained from ELT selection with their copy numbers within the selection.).In order to confirm biochemical activity of this chemical series compounds 1 and 2 (Figure 1c) were synthesized without the pendent DNA tags (see Supporting Information) as representatives of the selected feature for further evaluation. Confirmation of Biochemical Inhibition of UppS. Putative UppS binding compounds 1 and 2 identified via ELT screening were tested for their ability to inhibit the biochemical activity of S. aureus UppS. The assay11, 23 measures pyrophosphate produced from the condensation of IPP to the growing prenyl chain. The IC50 values of 1 and 2 were determined to be 190 ± 10 nM and 970 ± 90 nM, respectively, confirming that the binding selected for during the ELT screen resulted in productive enzymatic inhibition. Subsequent testing of 1 and 2 for activity against recombinant UppS enzyme derived from the important Gram-negative pathogens Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa showed no inhibition of these UppS proteins (IC50s > 100 µM in all cases, data not shown). Comparison of the amino acid sequences among various bacterial species show that side chains lining the hydrophobic channel are not as conserved as in the pyrophosphate region of the

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active site (see Figure S2), so it is not surprising that limited activity was observed across the species of bacteria tested. It is known in the literature that some classes of UppS inhibitors appear to have good cross-species activity10 at the enzyme level while others do not,9 with the differences likely being compound series dependent.

TM

Figure 1. Results from ELT selections. (a) Spotfire cube view of all compounds enriched with ≥ 2 independent DNA sequences. Axes represent building blocks (BB) used in cycle 1, 2, and 3, see Figure 1b. (b) Chemical structure of the main feature (circled in 1a) identified with enriched building blocks. (c) Chemical structures of UppS inhibitors 1 and 2 as synthesized without DNA tags.

In addition to the biochemical assay data, thermal shift was used to characterize the interaction of 1 and 2 with the S. aureus UppS protein. The Tm of S. aureus UppS protein alone was 52.8ºC. In the presence of 10 µM inhibitor, the Tm showed a significant shift to higher temperature. The change in Tm was observed to be +8ºC and +11ºC with compounds 1 and 2, respectively which is consistent with protein stabilization upon compound binding24. Antibacterial activity and mode of action studies. Compounds 1 and 2 were tested for antibacterial activity against a panel of bacterial pathogens (Table 1). Compound 1 has moderate antibacterial activity against two strains of S. aureus, including one methicillin-resistant S. aureus (MRSA) with a minimal inhibitory concentration (MIC) of 8 µg/mL and weak antibacterial activity against other Gram-positive pathogens with MIC of 64 – 128 µg/mL. Modest antibacterial activity (8-16 µg/mL) was obtained in M. catarrhalis and in H. influenzae when the major efflux system was deleted; however, even with efflux deficient mutants, no activity was obtained for other Gram-negative pathogens. Compound 2 has weak antibacterial activity against S. aureus (64 µg/mL) and low, if any, activity against other species tested. The relative antibacterial activity of 1 and 2 correlate well with their in vitro potencies against S. aureus UppS as does the lack of inhibition against several Gram-negative pathogens. Table 1. Susceptibility data for 1 and 2 vs. Grampositive and –negative bacteria


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Journal of Medicinal Chemistry MIC (µg/mL)

Bacterial strain

1

2

Cipro

a

Mero

b

Gram-positive pathogens S.aureus OXFORD

8

32-64

0.125

0.125

S.aureus WCUH29

4-8

32-64

0.5

2

S.pneumoniae 1629

32-64

128

0.25

0.016

S.pneumoniae Ery2

32-64

128

2

0.016

S.pyogenes 1307006P

64

128

0.5

128

128

128

>128

128

>128

128

>128

>8

0.5

a

Ciprofloxacin.

b

Meropenem.

Defining the antimicrobial mechanism of action of a small molecule is a critical part of hit and target validation, and demonstration of target-specificity is fundamental for the progression of a potential drug candidate. Not all compounds with antibacterial activity act through a target-specific mechanism and many instead act nonspecifically as alkylating agents, DNA intercalators, and detergents25. Indeed, very often hits from target-based screens, despite having biochemical activity, do not inhibit bacterial growth through target inhibition but through one or more of the above ‘nuisance’ mechanisms. Convincing demonstration of target-specific bioactivity greatly enables downstream optimization to maximize antibacterial activity while reducing off-target toxicity26. For example, antibacterial tetramic acids have been shown to be inhibitors of both UppS14 and bacterial RNA polymerase.27 Convincing mode of action data connect the observed in vitro biochemical inhibition with the antibacterial effect for compounds 1 and 2, was generated to justify subsequent medicinal chemistry optimization of this series. Consequently, compounds 1 and 2 were tested against S. aureus RN4220 carrying a plasmid (pYH4-UppS) which could be induced by anhydrotetracycline (ATc) to overexpress UppS protein.28,29 The MIC of 1 RN220 containing pYH4-UppS increased 4-fold relative to the parent RN4220 strain containing the empty plasmid in the absence of the ATc inducer, likely due to the leaky expression and/or pYH4-UppS plasmid copy number (Table 2). In the presence of 0.1 µg/mL ATc inducer, the MIC of 1 showed a further 2-fold increase over the un-induced system when compared to the plasmid vector control (Table 2). Similarly, an MIC increase was consistently observed for 2 against the S. aureus UppS over-expresser strain, but not the control. Although these MIC shifts are modest,

these data are consistent with the whole cell antibacterial activity of 1 and 2 being mediated through inhibition of UppS. To further confirm the antibacterial mode-of-action and obtain insight into the binding mode of the inhibitors, spontaneously arising resistant mutants of S. aureus were isolated on agar plates containing 32 µg/ml (4 x MIC) of compound 1 (see Supporting Information) and their uppS genes were sequenced. Two of the resistant mutants contained alterations in UppS (Phe103Leu and Phe148Leu) both of which confer an 8-fold MIC increase against 1 relative to wild type parent (Table 2) and have no effect on the MICs of other antibiotics or biocides tested (Table S2). This combination of overexpresser data and genetic data clearly demonstrate the on-target antibacterial activity of these UppS inhibitors. Crystal Structure of Compound 1 bound to UppS. In order to understand the details of the interaction between the pyrazole inhibitors and UppS, we determined crystal structures of S. pneumoniae UppS in complex with substrates and inhibitors. Similar to other UppS structures, S. pneumoniae UppS is a homodimer with an α+β fold, and the active site has two adjacent pockets where the substrates FPP and IPP bind (Figure S2). In the complex of S. pneumoniae UppS bound to FPP, the pyrophosphate moiety of FPP binds at the N-terminal helix formed by residues 28-32 and directly interacts with Arg41 and Arg79 (similar to E. coli UppS structure 1X068), while the farnesyl tail binds into a long, deep cavity lined by several hydrophobic side chains (Figure 2a). In the structure of S. pneumoniae UppS in complex with pyrazole 1, the inhibitor makes mostly hydrophobic interactions and is bound near the base of the hydrophobic FPP binding site, over 7Å from the catalytic region (Figure 2b). Compound 1 makes no direct hydrogen bonds with the protein; however, π stacking interactions are observed between the benzyl-isopropyl ether moiety and Phe143 and also between the benzothiophene moiety and both Phe94 and the γ-carbon of Met49. Compound 2, which lacks the aromatic thiophene ring, cannot make this latter π stacking interaction, which provides a structural rationale for the reduced enzymatic potency. The nature of the binding pocket and the ligand’s hydrophobic interaction at the binding site are also supported by ELT selection of BB3 primarily being hydrophobic aromatic moieties. To help rationalize the higher activity of compounds 1 and 2 against S. aureus relative to S. pneumoniae (Table 1), a published structure of the S. aureus UppS enzyme10 (blue ribbons) in complex with FPP was superposed with the S. pneumoniae structure (gray ribbons) in complex with 1 (Figure 2c). There are only two residue differences between S. pneumoniae and S. aureus UppS that are in direct proximity to the binding sites of 1, 2, or FPP, namely Leu126 (S. pneumoniae) Ile131 (S. aureus) and in direct proximity to the binding sites of 1, 2, or FPP, namely Leu126 (S. pneumoniae) Ile131 (S. aureus) and Leu145Ile150. The reduced conformational flexibility of Ile, relative to Leu due to branching at the beta carbon, likely provides a more stable hydrophobic surface against



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Table 2. Antibacterial activity of UppS inhibitors against UppS over-expresser and resistant strains a

MIC (µg/mL) vs. S. aureus RN4220 +pYH4 1 2

MIC (µg/mL) vs. S. aureus

S. aureus

+pYH4-UppS

+ATc

-ATc

+ATc

UppS WT

8

8

32

64

8

32

128

>128

Mammalian

UppS IC50 Cytotoxicity

-ATc 64

a

nd

c

e

a

UppS F103L

d

UppS F148L

d

(µM)

(µM)

64

64

0.19

25

nd

nd

0.97

100

b

b

MICs were the average of two independent determinations. Mammalian cytoxocity was determined in L5178y TK+/c d suspension cells, see Supporting Information. Wild-type, WT laboratory strain RN220. Resistant Mutants isolated e from WT laboratory strain RN220 in the presence of compound 1. nd = not determined.

which the ligand can pack. This notion is supported by the lack of electron density of the Leu145 sidechain in S. pneumoniae UppS such that it cannot be fully resolved. Superposition of the published S. aureus UppS structure overlayed with the co-crystal pose of 1 in S. pneumoniae UppS (Figure 2d) also provides a basis for rationalizing the resistant mutation data (Table 3). In the S. aureus structure, the two Phe residues (Phe103, Phe148) against which compound 1-resistant mutants arose lie along the hydrophobic product tunnel, beneath the bound inhibitor, distant from the FPP and IPP sites. Comparison of the amino acid sequences among various bacterial species show that side chains lining the hydrophobic channel are not as conserved as in the pyrophosphate region of the active site, so it is not surprising that limited activity was observed across the species of bacteria tested. Phe99 (Fig 2d, blue) sits edgewise between Phe103 and Phe148; producing a network of π stacking interactions that form a hydrophobic floor on which compound 1 directly resides. Both the benzyl-isopropyl ether (magenta) and benzothiophene (green) moieties are oriented edgewise about 4Å above the Phe148 phenyl and likely acquire affinity through π stacking. This provides a simple rationale for resistance against the Leu mutant, where these interactions would be dramatically compromised. The structural rationale for resistance to Phe103Leu is less straightforward and likely conformational. Resistance may arise due to disruption of the hydrophobic floor’s binding pocket. Additionally, the structure indicates the γ-carbon of Met54 (red) makes a πbonding interaction with the benzothiophene moiety of 1. Perturbation of the Met54 side chain via Phe103Leu mutation could substantially diminish this interaction. The location and identities of the observed compound 1resistant mutants corroborates that the antibacterial activity in S. aureus is mediated via UppS inhibition. Finally, selectivity of these compounds for bacteria over mammalian cells was evaluated using a mouse lymphoma cell line (L5178Y TK+/-) for inhibition of cell proliferation. Compounds 1 and 2 have TC50 of 25 and 100 µM (Table 2), respectively, suggesting a potential liability for mammalian cytotoxicity. Encouraged by the clear mode of action data and supported by structural information a small optimization effort was initiated on this pyrazole template. Initial goals were to improve biochemical activity against S. aureus and potentially broaden the spectrum of the series to other Gram-positive organisms. A strategy was to

extend the molecule to interact directly with the pyrophosphate binding region. This was thought to be useful both to decrease the lipophilicity of the inhibitors and to provide an anchor for binding in the pocket. Unfortunately, only very modest structural changes were tolerated and we had no success in modifying the template to reach the pyrophosphate binding region of the protein. In addition, we were unable to truncate the molecules and retain biochemical activity. It seems likely that the lack of orienting polar interactions between inhibitor and the UppS protein may contribute to the apparent poor optimizability of the current template. The combination of limited SAR, large size and very hydrophobic (clogP >5) character led us to discontinue optimization efforts on this series. Despite this, compounds 1 and 2 have proved valuable tool compounds for antibacterial target validation and intracellular target inhibition studies of UppS. CONCLUSION S. aureus UppS inhibitors have been identified and characterized from an Encoded Library Technology (ELT) screen. Employing a combination of techniques including in vitro biochemical inhibition, crystallography, and bacterial genetics, two substituted pyrazole inhibitors (1 and 2) with modest antibacterial activity were identified and their antimicrobial activity was demonstrably the result of UppS inhibition. Successful co-crystallization S. pneumoniae UppS characterized their binding to UppS in the FPP site, primarily making hydrophobic contacts with the protein. This hydrophobic FPP pocket provides a promiscuous site for small molecule binding, though it is more likely that successful molecules which target UppS will interact with the catalytic site in addition to the hydrophobic site. Through these studies we have confirmed that UppS is a validated and druggable antibacterial target, although the tractability of the current chemical series appears limited.

EXPERIMENTAL N-(3-Amino-3-oxopropyl)-1-benzyl-5-iodo-N-(4isopropoxybenzyl)-1H-pyrazole-4-carboxamide. (4Isopropoxyphenyl)methanamine (50 mg, 0.3 mmol) and acrylamide (25.8 mg, 0.36 mmol) in 2mL ethanol was added DIPEA (0.2 mL, 4 eq) and stirred at 70 oC overnight. The solvent was removed by vacuum and the residue was added to a solution of 1-benzyl-5-iodo-1Hpyrazole-4-carboxylic acid (99 mg, 0.3 mmol), HATU (115 mg, 0.3 mmol) and DIPEA (0.2 mL) in 2 mL DMF. The


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Figure 2. (a) X-ray structure of S. pneumoniae UppS with FPP. The left insert is a magnified view of the FPP pyrophosphate binding region. The right insert shows the farnesyl tail binds into a long, hydrophobic tunnel. Additionally, residues 79-93 in the FPP complex are displaced from 1.8Å up to 5.0Å compared to the native structure (apo helix shown in brown). (b) X-ray structure showing the S. pneumoniae UppS active site with compound 1 bound in a hydrophobic pocket. As in the native UppS structure, the residues in loop 73-83 remain disordered upon compound 1 binding. The propylcarboxamide moiety that linked the molecule to the DNA-encoded tag in the ELT screen is solvent exposed as expected (green, coming out of the page). The electron density around the benzyl-isopropyl ether (magenta) is weak suggesting that, even though deep in the pocket, it is in a dynamic state. Compound 1 lies nearly 10Å from the catalytic metal. (c) Superposition of x-ray structures of S. pneumoniae UppS in complex with compound 1 (blue ribbons) and S. aureus UppS in complex with FPP (4H8E; gray ribbons). The two residue differences between S. pneumoniae and S. aureus UppS in direct proximity to the binding sites of compounds 1, 2, or FPP are shown. (d) X-ray structure of S. aureus UppS (4H8E), superposed with the binding mode of compound 1 in complex with S. pneumoniae UppS. The two residues for which resistance mutants were observed in S. aureus are shown in green (Phe103Leu and Phe148Leu). mixture was stirred at room temperature for 30min. Ethyl acetate was added to the reaction mixture and washed with saturated NaHCO3 aqueous solution and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was loaded on to a 12g silica gel column and purified using MeOH and DCM to afford the title compound. (120 mg, + 1 72%). Purity >90%. MS (ESI) m/z [M+1] = 546.83. H NMR (400 MHz, CDCl3) δ 7.61 (s, 1H), 7.34-7.20 (m, 5H), 7.05 and 7.03 (d, J=8Hz, 2H), 6.85 and 6.83 (d, J=8Hz, 2H), 6.60 (broad s, 1H), 5.80 (broad s, 1H), 5,42 (s, 2H), 4.58-4.49(m, 3H), 3.69 (broad s, 2H), 2.62 (broad s, 2H), 1.33 and 1.32 (d, J=4Hz, 6H).

+

HRMS (M+H) calcd for [C24H27IN4O3 + H] 547.1201; found 547.1211. N-(3-Amino-3-oxopropyl)-5-(benzo[b]thiophen-5-yl)-1benzyl-N-(4-isopropoxybenzyl)-1H-pyrazole-4carboxamide (1). N-(3-Amino-3-oxopropyl)-1-benzyl-5iodo-N-(4-isopropoxybenzyl)-1H-pyrazole-4-carboxamide (30 mg, 0.055 mmol) and 2-(benzo[b]thiophen-5-yl)-4,4,5,5tetramethyl-1,3,2-dioxaborolane (29 mg, 0.11 mmol) in 1mL 1,4-dioxane was added Pd(PPh3)4 (3 mg, 2.75 µmol) and 0.3 mL 2M Na2CO3 aqueous solution and the mixture was stirred o at 80 C for 1hr. The mixture was purified with preparative



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HPLC to afford compound 3 (9 mg, 30%). HPLC Rt = 15min, + 1 purity>95%. MS (ESI) m/z [M+1] = 553.34. H NMR (400 MHz, CDCl3) δ 7.92 and 7.90 (d, J=8Hz, 1H), 7.71and 7.68 (d, J=12Hz, 2H), 7.54 and 7,52 (d, J=8Hz, 1H), 7.31-7.22( m, 5H), 7.03-7.00 (m, 2H), 6.87 and 6.85 (d, J=8Hz, 2H), 6.74 and 6.72 (d, J=8Hz, 2H), 6.49 (broad s, 1H), 5.64 (broad s, 1H), 5,26 (s, 2H), 4.49-4.41(m, 3H), 3.56 (broad s, 2H), 2.40 (broad s, 2H), 13 1.31 and 1.30 (d, J=4Hz, 6H). C NMR (100 MHz, CDCl3) δ 174.0, 166.8, 157.4, 140.7, 139.7, 137.9, 136.5, 128.7, 128.1, 128.0, 127.8, 127.1, 125.1, 124.9, 124.8, 123.9, 123.0, 116.0, 69.9, 53.4, + 52.8, 41.5, 33.9, 22.0; HRMS (M+H) calcd for [C32H32N4O3S + H] 553.2268; found 553.2270. N-(3-Amino-3-oxopropyl)-1-benzyl-N-(4isopropoxybenzyl)-5-(p-tolyl)-1H-pyrazole-4carboxamide (2). N-(3-Amino-3-oxopropyl)-1-benzyl-5iodo-N-(4-isopropoxybenzyl)-1H-pyrazole-4-carboxamide (30 mg, 0.055 mmol) and 4-methylbenezeboronic acid (15 mg, 0.11 mmol) in 1 ml 1,4-dioxane was added Pd(PPh3)4 (3 mg, 2.75 µmol) and 0.3 mL 2M Na2CO3 aqueous solution and the o mixture was stirred at 80 C for 1hr. The mixture was purified with preparative HPLC to afford compound 2 (12 mg, 43%). + HPLC Rt = 15min, purity>95%. MS (ESI) m/z [M+1] = 511.38. 1 H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 7.29-7.15 (m, 7H), 7.02 (m, 2H), 6.91 and 6.89 (d, J=8Hz, 2H), 6.78 and 6.76 (d, J=8Hz, 2H), 6.65 (broad s, 1H), 5.79 (broad s, 1H), 5,23 (s, 2H), 4.53-4.47(m, 1H), 4.37(s, 2H), 3.57 (broad s, 2H), 2.24 (broad 13 s, 2H), 2.40 (s, 3H),1.32 and 1.31 (d, J=4Hz, 6H). C NMR (100 MHz, CDCl3) δ 174.4, 166.9, 157.5, 139.7, 138.0, 136.5, 129.6, 129.4, 128.7, 128.3, 127.8, 127.1, 125.7, 116.1, 69.9, 53.3, 52.7, 41.4, + 33.9, 22.0, 21.4; HRMS (M+H) calcd for [C31H34N4O3 + H] 511.2704; found 511.2719.

AUTHOR INFORMATION Corresponding Author *RAS: e-mail: [email protected]. Phone: 610-9177163 or GE: e-mail: [email protected]. Phone: 781795-4423

ABBREVIATIONS USED UppS, Undecaprenyl pyrophosphate synthase; IPP, isopentenyl pyrophosphate; FPP, farnesyl pyrophosphate; UPP, undecaprenyl pyrophosphate; UP, undecaprenyl phosphate; ELT, Encoded Library Technology; SBP, Streptavidin Binding Protein; NTC, no-target control; BB, building block; MRSA, methicillin-resistant S. aureus; MIC, minimal inhibitory concentration; ATc, anhydrotetracycline.

ASSOCIATED CONTENT Supporting Information. Additional synthetic and experimental details. Final crystallographic coordinates are available in the RCSB PDB (5KH2 (S. pneumoniae UppS), 5KH4 (S. pneumoniae UppS complex with FPP), and 5KH5 (S. pneumoniae UppS complex with 1)). See Supporting Information for details. Authors will release the atomic coordinates an experimental data upon article publication.

REFERENCES 1. Ogura, K.; Koyama, T. Enzymatic aspects of isoprenoid chain elongation. Chem. Rev. 1998, 98, 1263-1276.

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