Structural and Biological Basis of Small Molecule Inhibition of

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Structural and biological basis of small molecule inhibition of Escherichia coli LpxD acyltransferase essential for lipopolysaccharide biosynthesis Xiaolei Ma, Ramadevi Prathapam, Charles Wartchow, Barbara Chie Leon, Chi-Min Ho, Javier de Vicente, Wooseok Han, Min Li, Yipin Lu, Savithri Ramurthy, Steven Shia, Micah Steffek, and Tsuyoshi Uehara ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00127 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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ACS Infectious Diseases

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Structural and biological basis of small molecule inhibition of Escherichia coli LpxD acyltransferase essential for lipopolysaccharide biosynthesis

Xiaolei Maa*, Ramadevi Prathapamb, Charles Wartchowa, Barbara Chie Leonc, Chi-Min Hoc†, Javier De Vicented†, Wooseok Hand†, Min Lic, Yipin Lue, Savithri Ramurthyd, Steven Shiaa†, Micah Steffeka†, Tsuyoshi Ueharab†*

aStructural

and Biophysical Chemistry, bInfectious Diseases, cProtein Sciences, dGlobal

Discovery Chemistry and eComputational Chemistry, Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, California 94608, USA.

* Correspondence should be addressed to [email protected] and [email protected]

†Present

addresses: CMH, University of California, Los Angeles, CA, USA; JDV, Denali Therapeutics, South San Francisco, CA, USA; WH, Novartis Institutes for BioMedical Research, Cambridge, MA, USA; SS, Gilead Sciences, Foster City, CA, USA; MS, Genentech, South San Francisco, CA, USA; TU, VenatoRx Pharmaceuticals, Malvern, PA, USA.


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LpxD, acyl-ACP-dependent N-acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound 1), that inhibit the enzymatic activity of E. coli LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target. We first found that compound 1 was oxidized in solution to the more stable aromatized tetrahydro-pyrazolo-quinolinone compound 1o. From the Escherichia coli strain deficient in efflux, we isolated a mutant that was less susceptible to compound 1o and had an lpxD missense mutation (Gly268Cys), supporting the cellular on-target activity. Using surface plasma resonance, we showed direct binding to E. coli LpxD for compound 1o and other reported LpxD inhibitors in vitro. Furthermore, we determined eight co-crystal structures of E. coli LpxD/inhibitor complexes. These co-structures pinpointed the 4ʹphosphopantetheine binding site as the common ligand binding hotspot, where hydrogen bonds to Gly269 and/or Gly287 were important for inhibitor binding. In addition, the LpxD/compound1o co-structure rationalized the reduced activity of compound 1o in the LpxDGly268Cys mutant. Moreover, we obtained the LpxD structure in complex with a previously reported LpxA/LpxD dual targeting peptide inhibitor, RJPXD33, providing structural rationale for the unique dual targeting properties of this peptide. Given that the active site residues of LpxD are conserved in multi-drug resistant Enterobacteriaceae, this work paves the way for future LpxD drug discovery efforts combating these Gramnegative pathogens.


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KEYWORDS Antibiotic discovery, Escherichia coli, lipopolysaccharide biosynthesis, LpxD, small molecule inhibition, on-target antibacterial activity, surface plasmon resonance, X-ray crystallography.



The outer membrane (OM) of Gram-negative bacteria provides a permeability barrier that, in concert with efflux pumps, serves to prevent the entry of toxic molecules into the cell1, 2.

Lipopolysaccharide (LPS) plays a major role in this barrier, and is composed of a lipid

A anchor that forms the outer leaflet of the OM, with a core oligosaccharide and covalently-linked repeating polysaccharide antigen that extends out from the cell surface3. Since the building block of LPS, Lipid A, is an essential structural component of most Gram-negative bacterial cells, disruption of lipid A biosynthesis is viewed as a promising strategy for the discovery of new antibacterials4. In particular, the identification of inhibitors of LpxC, the second enzyme of the pathway, has been the focal point of targetbased antibiotic discovery efforts4.

LpxD is the third enzyme in the lipid A biosynthesis pathway3 (Figure 1). The lpxD gene was initially identified as a firA allele in Escherichia coli. The E. coli firA mutation, firA200, eliminates the resistance to rifampicin associated with mutations of the target rpoB gene5. Similarly, a mutation in the gene highly homologous to firA in Salmonella typhimurium was found to render cells susceptible to antibiotics that normally do not penetrate through the OM6. Later, the firA gene product turned out to be the LpxD enzyme7, and reduction of LPS biosynthesis by the mutations increases cell permeability to antibiotics. Because the firA200 mutation displayed temperature-sensitive growth5, the lpxD gene was considered essential in E. coli. Consistent with this, attempts to make an lpxD deletion mutation in E. coli were not successful8-10. LpxD is also essential for growth in Pseudomonas aeruginosa9-11. Since some strains of Acinetobacter baumannii can grow without LPS12, 13, LpxD as well as LpxA and LpxC is dispensable for in vitro growth


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of these strains14. A. baumannii cells lacking LPS are resistant to colistin, but hypersusceptible to many other antibiotics, and are less virulent12-16. Therefore, inhibiting LpxD has direct antibacterial effects and the ability to potentiate drugs that have little activity against Gram-negative pathogens. In addition, the LpxD substrate accumulates in the lpxD temperature-sensitive mutant under non-permissive temperature and has been postulated to be toxic to the cell by acting like a detergent17. Together, these attributes make LpxD an especially attractive antibacterial target.

E. coli LpxD generates UDP-diacyl-GlcN by transferring R-3-hydroxymyristate from acyl-carrier-protein (ACP) to the free amine of UDP-3-O-(R-3-hydroxymyristyl)glucosamine (UDP-acyl-GlcN) (Figure 1). The LpxD reaction follows a compulsory ordered bi-substrate mechanism18, 19. The substrate R-3-hydroxymyristyl-ACP (acyl-ACP) binds LpxD first prior to the other substrate UDP-acyl-GlcN, followed by a transfer of the fatty acid moiety of acyl-ACP to the free amine of UDP-acyl-GlcN. The products, UDP2,3-diacyl-GlcN and holo-ACP, sequentially dissociate to regenerate LpxD9. Due to the complexity of the LpxD enzymatic reaction and the scarcity of purified LpxD substrates, drug discovery efforts on LpxD had been hampered by the lack of an enzymatic assay to enable small molecule screening. Despite the challenges, Jenkins and Dotson have published a series of seminal works to establish tool compounds for LpxD. They first employed the phage display method and discovered a peptide inhibitor RJPXD33 (TNLYMLPKWDIP-NH2)20 that simultaneously inhibits LpxD (KD = 6 µM) and LpxA (KD = 20 µM)20. Based on this peptide inhibitor, Jenkins describes in his doctoral dissertation21 the development of a fluorescent-based displacement method to screen for LpxD binders



in a library containing ~100,000 commercially-available chemicals and identify 11 small molecules that inhibited the LpxD reaction biochemically (Table 1). One of these, compound 1, has a half maximal inhibitory concentration (IC50) of 3.2 μM in the LpxD ThioGlo enzyme assay and a minimum inhibitory concentration (MIC) of 3.13 μg/mL against the E. coli ΔtolC strain21. Overexpression of LpxD in the sensitive strain reduces the compound’s antibacterial activity, implying LpxD inhibition as the primary mechanism of the antibacterial action in E. coli21.

Along with the advances in the identification of LpxD inhibitors, progress has also been made on the structural biology of LpxD. In recent years, the crystal structures of unliganded LpxD orthologs from Chlamydia trachomatis22, P. aeruginosa23 and A. baumannii24 as well as the first reported E. coli LpxD structure17 have become available. The structurally characterized LpxD proteins share moderate sequence conservation, namely 34%, 49%, and 37% sequence identities of C. trachomatis LpxD, P. aeruginosa LpxD, and A. baumannii LpxD to E. coli LpxD, respectively. Despite the relatively low homologies, the four LpxD orthologs share a similar sequential arrangement of secondary structure elements and possess the highly conserved fold topology, harboring an intertwined homotrimeric structure with a characteristic central prism of the left-handed βturn helix. By trapping various forms of ACP-LpxD complexes in crystal structures, Masoudi et al delineated the molecular mechanism of recognition of the ACP prosthetic group by E. coli LpxD19. The structures exhibit the 4ʹ-phosphopantetheine (4ʹ-ppt) site and two hydrophobic channels in the LpxD catalytic chamber. The hydrophobic N-channel and O-channel bind specifically to the acyl chains that are amide- and ester-linked to the


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UDP-acyl-GlcN lipid substrates, respectively. These works have provided detailed information on the three-dimensional structures and the catalytic mechanism of LpxD. In general, such information constitutes immense know-how for rational drug design. However, no co-structure has been reported to date for LpxD in complex with a small molecule or peptide inhibitor.

Here we report genetic, biophysical, and structural characterizations of small molecule inhibitors of E. coli LpxD reported by Jenkins et al21. We genetically validated the LpxD-specific antibacterial mechanism of action for compound 1o, the more stable aromatized compound 1. We then developed an in vitro binding assay using surface plasmon resonance (SPR) and detected compound binding to the E. coli apo-LpxD protein, providing biophysical evidence of direct binding for compound 1o and other reported scaffolds. Furthermore, we obtained eight LpxD/small molecule inhibitor cocrystal structures, for the first time revealing consensus ligand-binding hot spots. Lastly, we elucidated the crystal structure of the LpxD/RJPXD33 complex and revealed the dualinhibition mechanism by comparison to the published LpxA/RJPXD33 complex, providing important insights to the potential design of an LpxA/LpxD dual targeting small molecule inhibitor. Taken together, our work paves the way for future LpxD drug discovery efforts for combating multi-drug resistant Enterobacteriaceae, especially against carbapenemresistant E. coli and Klebsiella pneumoniae.



RESULTS AND DISCUSSION Rationales of the study Toward the development of an antibiotic targeting LpxD, we first evaluated whether any one of the eleven small molecule compounds reported as LpxD inhibitors21 could be a starting point for medicinal chemistry. The clogP values and the molecular weights (Table 1) of these molecules were high, and none of the compounds had the physicochemical properties that are suitable for development of antibiotics against Gram-negative bacteria25-27. Therefore, we did not pursue any of these compounds for optimization using medicinal chemistry. Instead, we attempted to utilize them as tool inhibitor molecules for characterization of LpxD as an antibacterial target. Since it was not known whether the reported inhibitors target LpxD directly, it was critical to validate the LpxD inhibition in orthogonal assays and to understand the mechanisms of inhibition. We obtained the same inhibitor compounds as those reported or close analogs when the original compounds were not commercially available (Table 1) and initiated the validation and characterization of these compounds. We first focused on compound 1 as it showed good solubility (120µM in PBS buffer) and had antibacterial activity against efflux-deficient E. coli.

Aromatization of compound 1 and 2 in solution We dissolved compound 1 (hexahydro-pyrazolo-quinolinones) and compound 2 (analog of compound 1) at 10 mM in DMSO and examined the compound identities using highthroughput LC-MS. The result showed different m/z by -2 from the values expected from the molecular weights of the compounds. This suggested that the compounds slowly oxidized to form the more stable tetrahydro-pyrazolo-quinolinones compound 1o and


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compound 2o (Table 1). This was further confirmed by synthesis of compound 1o and 2o by oxidation reaction of compound 1 and 2 with the oxidant 2,3-dichloro-5,6-dicyano-1,4benzoquinone (DDQ). Thus compound 1o and 2o were used hereafter for compound 1 and 2, respectively.

Mechanism of resistance to compound 1o in E. coli Compound 1o was not active against the wild type E. coli K-12 strain MG1655 (MIC >128 µg/mL), but inhibited the isogenic ΔtolC strain with an MIC of 1 µg/mL (Table 2), consistent with the reported antibacterial activity21. If compound 1o inhibits LpxD, overexpression of this target protein would reduce susceptibility to the compound through a titration effect. To test this hypothesis, we overexpressed the essential lpx genes in the lipid A biosynthesis pathway (lpxA, lpxB, lpxC, lpxD, lpxH, and lpxK) individually in the E. coli ΔtolC strain and determined the effects on susceptibility to compound 1o and the wellcharacterized LpxC inhibitor CHIR-090. As expected, CHIR-090 became 32-fold less potent in cells overexpressing LpxC (Table 2). Interestingly, LpxA overexpression also reduced CHIR-090 activity by 4-fold. LpxD overexpression increased the MIC of compound 1o by 8-fold, whereas no other overexpression strain examined had an effect on susceptibility.

Using a zone of inhibition method, we isolated two mutants (TUP0042 and TUP0074) that were less susceptible to compound 1o in the clear growth inhibition area of the E. coli ΔtolC strain. The single colony of TUP0042 showed a 128-fold increase in MIC of the compound and TUP0074 showed a 32-fold increase in MIC (Table 2). To



identify the mutations that might be conferring reduced susceptibility, we focused on the gene cluster including lpxD, lpxA, and fabZ. PCR-amplification and sequencing of the lpxD-lpxA-fabZ locus revealed that both mutants had changes in fabZ (Table 2). FabZ is a dehydratase required for fatty acid biosynthesis. Mutations in fabZ are known to suppress temperature-sensitive growth of lpxA and lpxC mutants28, 29 and to reduce the activity of LpxC inhibitors including CHIR-09030-32. One of the fabZ mutations identified, Pro56Leu, was reported to reduce susceptibility to the LpxC inhibitor B-78484 by 16-fold30. In addition, FabZ variants that reduce susceptibility to LpxC inhibitors are known to have reduced enzymatic activity, which serves to maintain a balance between biosynthesis of lipid A and fatty acids for growth in the presence of an LpxC inhibitor32, 33. Consistent with this, both of the isolated fabZ mutants were less susceptible to the LpxC inhibitor CHIR090. These results suggest that the fabZ mutations decrease susceptibility to compound 1o by a similar mechanism.

Having identified fabZ mutations as determinants of reduced sensitivity to compound 1o, we devised a strategy to minimize their selection. We carried out mutant selection using TUP0124, a strain expressing fabZ from an inducible multi-copy plasmid as well as the intact genomic locus. Under these conditions, the emergence of mutants with reduced FabZ activity should occur at a lower frequency. After incubation of the selection agar for 72 hours, a colony grew in a clear zone around the spot of compound 1o. The isolated mutant (TUP0043) was 8-fold less susceptible to compound 1o (Table 2). Sequencing of the chromosomal lpxD-fabZ-lpxA region identified no mutation in fabZ and a missense mutation in lpxD that encoded a Gly268Cys substitution. The lpxD mutant


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gene was then cloned in a low copy plasmid and the LpxDG268C variant was expressed in the E. coli ΔtolC strain (TUP0125) to examine its effect on compound 1o activity. The results showed that expression of LpxDG268C reduced susceptibility to compound 1o by 128-fold (Table 2). This was a more marked change than seen for expression of wild-type LpxD (8-fold). Taken together, these results indicate that compound 1o targets LpxD in E. coli cells and the Gly 268 residue of LpxD is important for compound 1o activity.

Direct binding of compound 1o and other scaffolds to LpxD as measured by SPR To examine specific interactions of compound 1o with LpxD, we determined the binding affinity of this compound to apo-LpxD using Biacore SPR with immobilized C-terminal avitagged LpxD protein. The affinity of the interaction of compound 1o with LpxD was measured to be 0.08 µM with kinetic fitting of the data using a 1:1 protein:compound interaction model only (Figure 2A, B), since equilibrium was not attained during the injection phase of this experiment. Some minor deviations from the 1:1 fitting model were observed, particularly at higher concentrations. For compounds 3-8 or their close analogs, equilibrium was attained during the injection phase and therefore a steady-state model was applied for affinity determination, showing KD values in the micro molar range and indicating direct binding of these compounds to E. coli apo-LpxD (Table S4). These results also demonstrate that the newly developed SPR assay for LpxD was useful to validate direct binding of compounds to apo LpxD and to determine their binding affinities.

LpxD co-structures and consensus ligand binding hot spots



To gain further structural insights into this set of chemically diverse LpxD inhibitors, we attempted to obtain X-ray crystal structures of E. coli LpxD in complex with these compounds. The crystal structure of N-terminally His6-tagged E.coli LpxD has been reported by Bartling C, et al17. However, this published C2 crystal form is not suitable for the purpose of structure-based drug design (SBDD) because the active site residues of LpxD are directly involved in crystal packing interactions. To identify a SBDD-compatible LpxD crystallography system, we rescreened the crystallization conditions in the presence of compound 1o or 2o, and identified a new crystal form P3221. Intriguingly, in a 1.7Å structure of E. coli LpxD crystal, compound 1o was found in the active site pocket at the interface of two LpxD monomers by partially occupying the N-channel, 4ʹ-ppt site, and the O-channel (Figure 3A, B). The flat tetrahydro-pyrazolo-quinolinone core interacted with the NH of G269 (chain A) directly and the NH of G287 (chain A), G257 (chain B) and the backbone carbonyl of F183 (chain B) via water mediated interactions. Aside from this hydrogen bonding network between the core scaffold and LpxD, three hydrophobic substituents phenyl, chloro-phenyl, and gem-dimethyl groups decorating the tetrahydro-pyrazolo-quinolinone core formed nonpolar interactions with the hydrophobic patches in the LpxD N-channel and O-channel. The co-structure also provides reasonable explanation for the mechanism of resistance to compound 1o by a substitution of Gly268 to a bulkier cysteine side chain, causing steric hindrance of the inhibitor binding to LpxD. In the same vein, replacement of the chloro-phenyl with a smaller thiophene moiety in compound 2o very likely decreases the protein-ligand interaction, consistent with the 10fold reduction in binding affinity measured by SPR and the reduced antibacterial activity compared to compound 1o.


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Starting from the LpxD/compound 2o co-crystals, we developed a three-step displacement soaking protocol to generate co-structures for compounds of interest (Figure S1). In contrast to the reported C2 crystal form of the E. coli apo-LpxD structure, one out of the three LpxD active sites (chain A) in the P3221 crystal form was much more exposed to the solvent, providing a possibility to displace compound 2o with a different chemical matter. Using the displacement-soaking method, we successfully obtained six additional LpxD co-structures in the 1.4 - 2.0Å resolution range for the SPR-confirmed E. coli LpxD inhibitors (Table S5). Clear differences in the electron density were observed for these compounds, represented by ligand chain A in the final deposited structures. Interestingly, compound 2o (chain B and C) were still present in two out of the three LpxD active sites, since they were sandwiched between the biological trimer and the nonbiological symmetry mate, and therefore were not displaced during soaking.

Compound 3, the smallest and the most ligand efficient hit among the compounds with SPR-confirmed binding, interacted with the LpxD 4ʹ-ppt site and N-channel, but not with the O-channel (Figure 3C). The triazole and sulfonyl moieties functioned as hydrogen bond acceptors and engaged the main chain nitrogens of G287 and G269 of the monomer subunit on the right (chain A), respectively. The NH group acted as a hydrogen bond donor and interacted with N274 towards the monomer subunit on the left (chain B). Ethoxyphenyl and the o-tolyl groups were directed towards the N-channel and surrounded by a number of non-polar amino acids. While compound 1o and compound 3 engaged both LpxD monomers by direct or water-mediated hydrogen bonds, compound



4.1, compound 5, compound 6, compound 7 and compound 8.1 bound at the interface of two LpxD monomers by preferentially forming hydrogen bonds to the G287 and/or G269 of the LpxD monomer on the right (chain A) (Figure 3D-H). No hydrogen bond was established between these compounds and the left LpxD monomer. Taken together, the same set of protein residues, G269 and G287, was commonly involved in the recognition of these structurally diverse LpxD ligands. Since the 4ʹ-ppt site is used to interact with the acyl/4ʹ-ppt groups of acyl-ACP during the catalytic cycle19, the G269/G287 region of the 4ʹ-ppt site may represent a ligand binding “hot spot” that could be further exploited to improve the compound binding affinity.

Structural basis for the LpxA/LpxD dual targeting peptide RJPXD33 The antimicrobial peptide RJPXD33 inhibits both E. coli LpxD and LpxA acyltransferases by competing with the acyl-ACP substrate20. Although the molecular mechanism by which RJPXD33 interacts with E. coli LpxA is well understood34, attempts to co-crystallize LpxD with RJPXD33 were unsuccessful both in our hands and in previous studies34. With the success of the displacement-soaking method for small molecule co-structures, we attempted this method with FITC-labeled RJPXD33. Indeed, we were able to gather a 2.1Å dataset that allowed us to determine the co-structure of E. coli LpxD/RJPXD33. Importantly, only five residues (LYMLP) of RJPXD33 (TNLYMLPKWDIP-NH2) were well defined in the crystal structure, as shown in the omit electron density map (Figure S2). The two N-terminal residues of RJPXD33 (TN) and the five C-terminal residues (KWDIP) could not be modelled as they protruded into the solvent. In contrast to the proposed binding model of RJPXD33-LpxD by Jenkins and Dotson34, our co-crystal structure


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revealed an unforeseen interaction mode. When bound to E. coli LpxD, RJPXD33 adopted a type I two-residue beta-hairpin conformation, which was stabilized by an internal hydrogen bond between the carbonyl of Leu3 (i) and the NH group of Leu6 (i+3) (Figure 4A and B). The main chain carbonyls of the central two residues, Tyr4 and Met5, in this short beta-hairpin of RJPXD33 engaged the main chain NH groups of Gly287 and Gly269. Furthermore, the side chains of Tyr4 and Met5 were projected into the LpxD Nchannel and formed nonpolar interactions. The side chains of Leu3 and Leu6 were packed in the hydrophobic patch of the C-channel while Pro7 of RJPXD33 was buttressed by LpxD Phe183.

A side-by-side structural comparison of LpxD/RJPXD33 and LpxA/RJPXD33 showed that the trajectory and the conformation of the RJPXD33 peptide are quite distinct in the two structures (Figure 4C and D). When bound to LpxD, RJPXD33 formed a betahairpin that is perpendicular to the LpxD 3-fold symmetry axis. When bound to LpxA, RJPXD33 adopts a much more extended, linear conformation that runs parallel to the LpxA 3-fold symmetry axis34. An earlier report showed that the deletion of the C-terminal six residues of RJPXD33 has a much greater impact on affinity for LpxD than LpxA34, which is consistent with our structural observation that one of those six residues contributes to LpxD binding while none are involved in LpxA binding34. These results indicate that the conformational versatility of RJPXD33 peptide is an important structural feature that allows the same peptide to interact with diversified acyltransferases LpxD and LpxA. In general, peptide inhibitors targeting a cytoplasmic protein are not considered as a starting point for medicinal chemistry due to the lack of the peptide delivery system



into the bacterial cytoplasm. Thus, it is necessary to design a small molecule compound that targets the two essential acyltransferases or either one of them in the lipid A biosynthesis pathway. Our structural information on the binding of the tool compounds and the peptide to LpxD will help the design of such small molecule inhibitors for antibiotic development.

CONCLUSIONS In this report, we employed chemical biology, molecular biophysics, and structural biology in the characterization of LpxD small molecule inhibitors reported previously21. We first showed aromatization of compound 1 and 2 leading to the more stable oxidized compound 1o and 2o. Our genetic studies using overexpression and spontaneous mutant selection indicated that the antibacterial activity of compound 1o against efflux-deficient E. coli is mediated by blockage of LPS synthesis specifically through inhibition of LpxD. We next developed an SPR assay and showed the direct binding of compound 1o to E. coli apo-LpxD. We then structurally characterized these LpxD binders by establishing a three-step displacement soaking protocol. Using this method, we successfully obtained co-crystal structures of ten chemically diverse scaffolds, providing mechanistic insights into LpxD inhibitors. Compound 1o bound specifically to the LpxD catalytic site by occupying the 4ʹ-ppt site/N-channel/O-channel, thus preventing LpxD from interacting with both substrates acyl-ACP and acyl-UDP-GlcN. Moreover, these co-crystal structures indicate the 4ʹ-ppt site as a common ligand binding hot spot and revealed additional key interacting moieties that could set the stage for pharmacophore-based virtual screening.


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This is the first time where ligands binding to LpxD have been structurally depicted, and we believe that this knowledge will significantly aid in the design of novel inhibitors with improved potency.

MATERIALS AND METHODS Chemistry Compounds 1o and 2o were synthesized and evaluated for this work. All other compounds were purchased from eMolecules. Compounds 1, 1o, 2 and 2o were characterized using LC-MS and NMR (described in the Supporting Information).

Synthesis of compounds 1o: To a solution of compound 1, 4-(2-chlorophenyl)-3-hydroxy7,7-dimethyl-2-phenyl-6,7,8,9-tetrahydro-2H-pyrazolo[3,4-b]quinolin-5(4H)-one, (258 mg, 0.614 mmol) in dioxane (6 mL) was added DDQ (139 mg, 0.614 mmol) at room temperature. The reaction mixture was stirred for 16 h. After quenched with saturated sodium carbonate solution, the mixture was extracted with EOAc. The organic layer was washed with water and brine, dried over anhydrous sodium sulfate, filtered off, and concentrated in vacuo. The crude product was purified by reversed phase HPLC to yield compound

1o,

4-(2-chlorophenyl)-3-hydroxy-7,7-dimethyl-2-phenyl-7,8-dihydro-2H-

pyrazolo[3,4-b]quinolin-5(6H)-one, as its trifluoroacetic acid (TFA) salt.

Synthesis of compounds 2o: Compound 2, 3-hydroxy-7,7-dimethyl-2-phenyl-4-(thiophen2-yl)-7,8-dihydro-2H-pyrazolo[3,4-b]quinolin-5(6H)-one, was prepared using the same



method to yield compound 2o, 3-hydroxy-7,7-dimethyl-2-phenyl-4-(thiophen-2-yl)-7,8dihydro-2H-pyrazolo[3,4-b]quinolin-5(6H)-one as its TFA salt.

Bacterial strains, plasmids, growth media, and antimicrobial susceptibility testing. Bacterial strains and plasmids used in this study are listed in Table S1. L-broth (Miller) and LB-agar were used for routine growth and bacterial selection during plasmid construction. To maintain pMMB-based plasmids, 10 µg/mL chloramphenicol was added to media. Mueller-Hinton II broth (cation adjusted) (MHBII) or Muller-Hinton agar (MHA) were used for growth for antimicrobial susceptibility testing or determination of MIC of compounds. MHBII supplemented with 100 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 10 µg/mL chloramphenicol was used for MIC determination of strains carrying plasmids. The detailed method of MIC determination is described in Supporting Information.

Construction of plasmids used for genetic studies. Primers used in this study are listed in Table S2. The PCR fragments were amplified using Phusion High-Fidelity DNA Polymerase with primers and templates as shown in Table S3. Amplified DNA fragments were purified and cloned into the EcoRI-HindIII site of pMMB206 vector using ligation or GeneArt cloning. After transformation of Top10 competent cells and selection on LB-agar containing 10 µg/mL chloramphenicol, colonies were screened with PCR-amplification followed by DNA sequencing using primers TU12 and TU96.


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Isolation of mutants less susceptible to compound 1o and targeted sequencing. The E. coli ΔtolC strain was grown on MHA overnight at 37°C. Several colonies were picked using a BBL-prompt and resuspended to BBL saline tube (Becton Dickinson). One hundred microliters of the cell suspension of 1.5x108 colony forming units (CFU)/mL were spread on MHA. After the agar plate was dried at room temperature for 20 min, 1 µL of 12.8 mg/mL compound 1 stock solution in DMSO was spotted at the center of the plate. The plate was then incubated at 37°C for 72 hours. After overnight incubation, a clear zone around the spot was observed in a lawn of bacteria. Colonies appearing in the clear zone were purified by streaking them on MHA and a single colony was saved for susceptibility testing. To isolate resistant mutants from E. coli ΔtolC carrying a fabZ plasmid (TUP0124), a similar mutant selection experiment was performed on MHA containing 10 µg/mL chloramphenicol for plasmid maintenance and 100 µM IPTG to induce FabZ expression from the plasmid. The lpxD-fabZ-lpxA genetic region of the isolated mutants was PCR-amplified using primers TU117 and TU243. The amplified DNA was sequenced using the primers TU116, TU117, TU243, TU276 and TU332.

Construction of plasmids expressing N-terminal 6His-TEV-tagged EcLpxD and N6His-TEV-EcLpxD-Avi. DNA encoding EcLpxD 4-341 with an N-terminal 6His-TEV tag and Ser-Gly-Gly-Ala linker was synthesized and cloned into pET24a downstream of the T7 promoter. The Avi-tagged construct was engineered utilizing seamless cloning to introduce a BirA recognition sequence C-terminal to E. coli LpxD.



Expression and Purification of 6His-TEV-tagged EcLpxD. 6His-TEV-EcLpxD was expressed in the BL21(DE3) E. coli strain in 2x TB medium supplemented with trace metals and 50 µg/mL kanamycin. Cells containing the 6His-TEVEcLpxD expression plasmid were cultured to an OD600 of ~1 at 37oC before induced with 0.2 mM IPTG at 20°C overnight. Cells were harvested at 5,000 x g for 15 min and stored at -80°C. Frozen cells expressing 6His-TEV-EcLpxD were resuspended into Ni-A buffer (50 mM HEPES-NaOH (pH 8.0), 500 mM NaCl, 10% glycerol, 1 mM TCEP) containing two Promega Protease inhibitor cocktail tablets and 12,000 U Promega Universal nuclease per 40 mL of lysate. Resuspended cells were lysed via sonication on ice at 70% output for 1.5 min in 45 sec bursts. Supernatant was clarified by centrifugation at 50,000 x g for 30 min at 4°C. 6His-TEV-EcLpxD protein was captured on a His-Trap FF column, washed with Ni-A buffer containing 50 mM imidazole before eluted with Ni-A buffer containing 400 mM Imidazole. The 6His-TEV-EcLpxD protein was cleaved overnight at 4°C with 900 U TEV protease per 1 mg protein. Non-cleaved protein and TEV protease were removed by a His-Trap column before LpxD protein was purified by a Superdex 200 size exclusion column that had been equilibrated in 20 mM HEPES-NaOH (pH 8.0), 300 mM NaCl, 1mM TCEP. The final purified protein was concentrated up to 15 mg/mL and stored at -80°C.

Expression, purification and preparation of biotinylated EcLpxD-Avi. 6His-TEV-EcLpxD-Avi was co-transformed into the BL21(DE3) E. coli strain with pBirAcm carrying the birA gene and the chloramphenicol resistance gene (cat). Cells were grown in 2x TB medium supplemented with trace metals, 50 µg/mL kanamycin and 25 µg/mL


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chloramphenicol to an OD600 of 0.6-1.0 and then the protein was induced by addition of 1 mM IPTG for 4 hours at 37°C. The Avi-tagged EcLpxD protein was purified similarly to the above-mentioned purification procedure for the non-Avi-tagged construct except that the final buffer used for storage was 20 mM HEPES-NaOH (pH 7.5), 500 mM NaCl, 1 mM TCEP.

SPR binding assay. SPR binding studies for inhibitors of LpxD were performed on a Biacore T200 instrument at 20°C at a flow rate of 60 µL/min using a streptavidin-coated sensor chip (GE BR100531) to immobilize the C-terminal Avi-tagged LpxD protein to a final loading level of ~6000 RU. Compounds dissolved in DMSO were diluted by 50-fold in assay buffer to a final DMSO concentration of 2%. A typical assay uses a 7-point, 2-fold dilution series. The assay buffer was standard phosphate buffered saline containing 1 mM EDTA, 0.01% P20 detergent, and 1 mM TCEP. SPR data were analyzed with Biacore Evaluation Software v2.0 using a global 1:1 binding kinetic interaction model for compound 1o35 or steadystate model for all other compounds36.

Crystallography. E. coli full-length N-His-LpxD (15.7 mg/mL) was incubated with 2 mM compound 2o for 1 hour on ice prior to crystallization. LpxD/compound 2o co-crystals were grown at 18°C in 0.2 M Mg-formate (pH 7.0) containing 20% PEG3350 with a 1:1 ratio of protein to crystallant using the sitting-drop vapor diffusion method. Crystals typically appeared by day 2-3 and reached full size by day 5. Fresh (less than two weeks old) LpxD/compound



2o co-crystals were subsequently used for displacement-soaking to generate LpxD structures in complex with inhibitors of interest. The co-crystals were soaked in reservoir solution supplemented with 1-2 mM compound through three rounds of serial transfer over the time course of 1.5-2 days. Before data collection, crystals were cryoprotected in soaking buffer plus 20% glycerol and flash cooled in liquid nitrogen.

Data collection and structure determination. Data were collected on a single crystal cooled to 100 K using synchrotron radiation sources such as Advanced Light Source (ALS) beamline 5.0.1, 5.0.2, and Advanced Photon Source (APS) beamline 17-ID. Crystals of LpxD/inhibitor complexes typically diffracted to 1.5-2.5 Å resolution and belonged to the space group P3221 with unit cell parameters a=98.9 Å, b=98.9 Å, c=215.3 Å, α=90°, β=90°, γ=120°. The asymmetric unit contained one LpxD homotrimer in complex with two copies of compound 2o (not displaced) and one copy of compound of interest. Data were processed using autoPROC37. Data collection statistics from LpxD/inhibitor complexes are shown in Table S5. The structures of the E. coli LpxD/inhibitor complexes were solved at various resolution by molecular replacement with Phaser38 using the published crystal structure of the apo form of E. coli LpxD17 (PDB ID code: 3EH0) as the starting model. The compound of interest was first placed by the rhofit39 and further refined in Phenix40. Subsequent rounds of model building and refinement with the Phenix.refine program were carried out until convergence.

Accession numbers:


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Coordinates and structure factors were deposited in the Protein Data Bank. The accession codes of E. coli LpxD in complex with inhibitors were with compound 1o (PDB 6P83), compound 2o (PDB 6P84), compound 3 (PDB 6P85), compound 4.1 (PDB 6P86), compound 5 (PDB 6P87), compound 6 (PDB 6P88), compound 7 (PDB 6P89), compound 8.1 (PDB 6PA), and FITC-RJPXD333 (PDB 6P8B).



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Table 1. E. coli LpxD inhibitors Structure

Compound name (eMolecules catalog number) Compound 1 (6359-0284)

MW

cLogP

419.9

5.5

Structure

Compound name (eMolecules catalog number) Compound 4.1

406

3.7

364.4

2.5

377.4

3.6

431.5

4.0

396.5

2.7

N

O O HN

Compound 1o

417.9

6.2

Compound 5 (D391-1003)

H N

Cl OH

N

N N

N

3.8

N

N

O

474.6 O

N

N H

MW

N

Cl OH

O

clogP

S

O

O O

Compound 2 (6359-0289)

391.5 O

4.6

S OH

Compound 6 (3031-0919)

N

NH

O

Compound 2o

389.5 O

O

H N

O

N

N H

O O

5.7

S OH

Compound 7 (C672-0030)

O

N

N

N

N

N

HN O O O

Compound 3 (C200-4634)

358.4 N N N

O

NH2

3.5

O S O

Compound 8 (G284-0533)

O O O N

N

NH

N N

Compound 4 (G673-0639)

490.6

N N

N O

3.2

Compound 8.1

N N

O O

O

O

NH

N N

S

N

HN

Compound 4 and 8 were not obtained due to unavailability in the eMolecules compound library. Instead, the close analogs, compound 4.1 and 8.1, were obtained and used in the SPR assay.


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Table 2. Compound 1o MIC and effects on gene overexpression and mutations Compound 1 MIC (µg/mL) >128

CHIR-090 MIC (µg/mL) 0.25

ΔtolC

1

0.016

TUP0115

ΔtolC Plac::lacZα (control)

1

0.008

TUP0116

ΔtolC Plac::lpxA

1

0.032

TUP0118

ΔtolC Plac::lpxC

1

0.25

TUP0119

ΔtolC Plac::lpxD

8

0.008

TUP0120

ΔtolC Plac::lpxH

1

0.008

TUP0117

ΔtolC Plac::lpxB

1

0.008

TUP0121

ΔtolC Plac::lpxK

1

0.008

TUP0042

ΔtolC fabZ [FabZCys139Tyr]

128

0.25

TUP0074

ΔtolC fabZ [FabZPro139Leu]

32

0.25

TUP0124

ΔtolC Plac::fabZ

1

ND

TUP0043

ΔtolC lpxD [LpxDGly268Cys]

8

ND

128

ND

Strain

Description

MG1655

Wild type

VECO2526

TUP0125 ΔtolC Plac::lpxD [LpxDGly268Cys] ND: not determined



DATA AVAILABILITY Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 6P83 (compound 1o), 6P84 (compound 2o), 6P85 (compound 3), 6P86 (compound 4.1), 6P87 (compound 5), 6P88 (compound 6), 6P89 (compound 7), 6P8A (compound 8.1), and 6P8B (peptide RJPXD33). The datasets are available from the corresponding authors on reasonable request.

SUPPLEMENTAL MATERIAL The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Characterization of compounds 1, 1o, 2 and 2o by LC-MS and NMR; antimicrobial susceptibility testing; Bacterial strains, primers and templates; SPR KD values determined for reported E. coli LpxD inhibitors; Data collection and refinement statistics; displacement soaking protocol; and the 2Fo − Fc electron density map of RJPXD33.

ACKNOWLEDGEMENTS We thank the staff of Sector 5 at the Advanced Light Source, the staff of IMCA-CAT 17ID at Advanced Photon Source and the staff of Crystallographic Consulting, LLC, for assistance in collecting X-ray crystallographic data. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. This research


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used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We also thank Mina Mostafavi for constructing the pMM6 plasmid, Catherine Jones for editorial assistance, and Don Ganem, Jennifer Leeds, Heinz Moser, Isabel Zaror, Dirksen Bussiere, and past and present colleagues in NIBR Emeryville, CA for support and discussion.

AUTHOR CONTRIBUTIONS JD, WH, SR, and YL synthesized or acquired compounds used in this work. PR and TU designed and conducted bacterial genetics studies. MH, BC, and ML performed cloning, protein expression and purification. MS and CW carried out the SPR studies. SS and XM performed crystallography and structural analysis. XM, RP, CW, BL, WH, and TU wrote the manuscript.



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FIGURE LEGENDS Figure 1. The role of LpxD in the Kdo2-lipid A biosynthesis pathway LpxD is the third enzyme of the pathway in E. coli and catalyzes the transfer of R-3hydroxymyristate (purple) from ACP to the free amine of UDP-3-O-acyl-glucosamine, generating UDP-diacylglucosamine.

Figure 2. BIAcore SPR binding analyses of compound 1o binding to E. coli apoLpxD (A) SPR sensorgram of the titration of compound 1o on C-avi-tagged LpxD immobilized on a streptavidin-coated sensor chip. Sensorgram was obtained by using a different concentration of ligands (colors represent 2x-dilution series, ranging from 6.4 µM to 0.1 µM). (B) The binding curves for the interaction of compound 1o with LpxD were fit using a 1:1 kinetic model to generate a KD of 0.08±0.04 μM (N=3). The black lines show the fitting curves.

Figure 3. Crystal structures of LpxD in complex with inhibitors and substrate acyl-ACP LpxD is shown as ball and sticks with carbon colored orange for protomer A, light yellow for protomer B, oxygen colored red and nitrogen colored blue. The active site pocket is at the interface between protomer A and B. The LpxD active site pocket surface is represented as aromatic lipophilic (white), non-aromatic other (mostly aliphatic)



lipophilic surface (green), hydrogen bonding acceptor potential (red), and hydrogen bond donor potential (blue). (A) LpxD/acyl-ACP complex reported previously19. The 4ʹppt group and the two β-OH-C14 acyl chains are shown as ball and sticks with carbon colored in green and light/dark green. (B-H) LpxD structures in complex with compound 1o, compound 2o, compound 3, compound 4.1, compound 5, compound 6, compound 7, and compound 8.1 are shown as ball and sticks with carbon colored purple, dark green, deep blue, cyan, light green, salmon and dark red, respectively.

Figure 4. LpxD structure in complex with the peptide inhibitor RJPXD33 (TNLYMLPKWDIP-NH2), compared with LpxA/RJPXD33 complex (A,B) 2.1Å structure of LpxD bound to FITC-RJPXD33 (LYMLP visible). (A) LpxD and active site pocket surface representation as shown in Figure 3. (B) Detailed interactions between LpxD and the structured region of RJPXD33. Carbon is shown as ball and stick in deep green. Hydrogen bonds are shown as dashes in deep blue. (C,D) Previously reported 1.9Å structure of E. coli LpxA bound to RJPXD3334 (TNLYML visible) (PDB ID: 4J09). (C) LpxA is shown as ball and sticks with carbon colored orange for protomer A, light yellow for protomer B, oxygen colored red and nitrogen colored blue. The active site pocket surface is represented as shown in (A). (D) Detailed interactions between LpxA and the structured region of RJPXD33 peptide, shown as ball and sticks with carbon colored light green. Hydrogen bonds are shown as dashes in deep blue.


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