Different Poses for Ligand and Chaperone in Inhibitor Bound Hsp90 and GRP94: Implications for Paralog-specific Drug Design

Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian hsp90 paralogs, which are cytoplasmic Hsp90α and β, ER GRP94, and mitochondrial Trap-1. Given that each of the hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each hsp90 paralog. Here we present side by side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-hsp90 inhibitors Geldanamycin and Radamide. These structures reveal paralog specific differences in the Hsp90 and GRP94 conformations in response to Geldanamycin binding. We also report significant variation in the pose and disparate binding affinities for the Geldanamycin-Radicicol chimera Radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors

Topoisomerase Inhibitors Addressing Fluoroquinolone Resistance in Gram-Negative Bacteria.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region

Structure-guided enzymology of the lipid A acyltransferase LpxM reveals a dual activity mechanism

Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and "reset" mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases' structure-mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins

The dependence of LpxH for growth is abrogated by inhibition of LpxC under standard laboratory conditions.

<p>(A) NB48062-JWK0133 was streaked on MHIIB agar supplemented with 1 mM IPTG and grown overnight at 37°C to induce <i>lpxH</i> expression. The following day, cells were washed repeatedly and resuspended to an OD₆₀₀ of 0.01, and 100 μL was plated on MHIIB plates without IPTG. Sterile filter discs containing IPTG, DMSO, or CHIR-090 were placed on the plates which were then incubated 37°C for 24 hours. Left panel; growth of NB48062-JWK0133 was not observed under non-inducing conditions (minus IPTG, DMSO). Center panel; growth of NB48062-JWK0133 is restored in the presence of IPTG. Right panel; NB48062-JWK0133 grew under non-inducing conditions in the presence of the LpxC inhibitor CHIR-090. (B) An overnight culture of NB48062-JWK0133 under inducing conditions (+ IPTG) was diluted to an OD₆₀₀ of 0.1 and then was diluted 100-fold into MHIIB containing 10% Alamar Blue. Next, 100 μL of the inoculum was added to the wells of a 96-well plate containing CHIR-090 to a final assay concentrations ranging from 0.25–64 μg/ml. The plate was incubated for 6 hours at 37°C before fluorescence reading (545ex (nm)– 590em (nm)) on the SpectraMax and analyzed with Softmax® Pro software v 5.4.1.</p

Depletion of LpxH causes accumulation of DSMP.

<p>(A) LCMS-MRM quantification of DSMP with three 12:0(3-OH) acyl groups and one 14:0(3-OH) acyl group and with two 12:0(3-OH) acyl groups and two 14:0(3-OH) acyl groups for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The experiment was performed in triplicate and bars show the mean value and SD (two-tailed student t-test *, P<0.05 and **, P<0.01) between NB46082-JWK0133 in the presence or absence of IPTG). Data shown was normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>].</p

Depletion of LpxH causes accumulation of UDP-Diacyl-GlcN (LpxH substrate).

<p>A) The LCMS-MRM quantification of lipid A precursor UDP-Diacyl-GlcN is shown for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The <i>m/z</i> [M–H⁺]^- for UDP-Diacyl-GlcN containing 2 acyl groups, 12:0(3-OH), 12:0(3-OH) is 960.5 and for UDP-Diacyl-GlcN with 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH) the <i>m/z</i> [M–H⁺]^- is 988.5. Experiments were performed in triplicate and bars show the mean value and SD (two-tailed student t-test, **, P<0.01) between NB46082-JWK0133 in the presence or absence of IPTG. Data shown were normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>]. B) Extracted ion chromatogram (EIC) of NB48062-JWK0133 in the presence or absence of IPTG for 2 acyl groups, 12:0(3-OH), 12:0(3-OH). C) Extracted ion chromatogram of NB48062-JWK0133 in the presence or absence of IPTG for 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH).</p

Transmission EM images of NB48062-JWK0133 plus or minus IPTG.

<p>LpxH depleted cells (-IPTG) display gross morphology changes. The experiment was repeated twice with similar results.</p

Schematic illustration of the <i>lpxH</i> regulated expression strain NB46082-JWK0133 and its dependence on IPTG induction for cell growth.

<p>(A) The chromosomal allele of <i>lpxH</i> is regulated by the P_tac promoter (inducible by IPTG). Plasmid pNOV108 provided extra copies of <i>lacI</i> to enhance repression of <i>lpxH</i> in the absence of IPTG. pNOV108 also contained <i>alaS</i> so that it could be maintained by complementation of an <i>alaS</i> deletion on the chromosome, eliminating the need for antibiotic selection. (B) Growth of NB48062-JWK0133 was IPTG dependent. C) Sub-culture growth curve of NB48062-JWK0133 plus or minus IPTG. Arrows indicate time points of sample collection for LCMS-MRM analysis, CFU determination, TEM images, RT-qPCR, and CHIR-090 rescue. D) A significant loss in viability (*, <i>P</i> ≤ 0.01) was observed at 1.5 OD₆₀₀ under LpxH depletion conditions (-IPTG) compared to inducing conditions (+IPTG).</p

Depletion of LpxH causes accumulation of an alternative LpxC product containing a C14:0(3-OH) acyl chain.

<p>The LCMS-MRM quantification of UDP-3-<i>O</i>-[(<i>R</i>)-3-OH-C_12/14]-GlcN for acyl group 12:0(3-OH) and for acyl group 14:0(3-OH) is shown for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The experiment was performed in triplicate and bars show the mean value and SD (two-tailed student t-test ***, P<0.001) between NB46082-JWK0133 in the presence or absence of IPTG. Data shown was normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>].</p

The monobactam LYS228: mode of action and mechanisms decreasing in vitro susceptibility of Escherichia coli and Klebsiella pneumoniae

The monobactam chemical scaffold is attractive for the development of new agents to treat infections caused by drug-resistant Gram-negative bacteria since it is stable to metallo-β-lactamases (MBLs). However, the clinically used monobactam aztreonam lacks stability to serine β-lactamases (SBLs) that are often co-expressed with MBLs. The novel monobactam LYS228 is stable to MBLs and most SBLs. LYS228 bound purified Escherichia coli penicillin binding protein 3 (PBP3) similarly to ATM (k2/Kd = 367504 s-1M-1 and 409229 s-1M-1, respectively) according to stopped-flow fluorimetry. A gel-based PBP binding assay showed that LYS228 bound mainly to E. coli PBP3, with weaker binding to PBP1a and PBP1b. Exposing E. coli cells to LYS228 caused filamentation, consistent with cell division defects resulting from inhibition of PBP3. No single-step mutants were selected from twelve Enterobacteriaceae strains expressing different classes of β-lactamases at 8X the minimum inhibitory concentration (MIC) of LYS228 (frequency <2.5x10-9). At 4X the MIC, mutants were selected from two of twelve strains at frequencies of 1.8x10-7 and 4.2x10-9. LYS228 MICs were ≤ 2 μg/mL against all mutants. These frequencies compared favorably to those obtained with meropenem and tigecycline. Mutations decreasing LYS228 susceptibility occurred in ramR and cpxA (Klebsiella pneumoniae) and baeS (E. coli and K. pneumoniae). Susceptibility of E. coli ATCC 25922 to LYS228 decreased 256-fold (MIC 0.125 to 32 µg/mL) after 20 serial passages. Mutants had accumulated mutations in ftsI (encoding the target, PBP3), baeR, acrD, envZ, sucB and rfaI. These results support the continued development of LYS228, which is currently undergoing Phase II clinical trials for complicated intraabdominal infection and complicated urinary tract infection (Clinicaltrials.gov NCT03354754)