Sandra Lightle

Discovery of Antibacterial Biotin Carboxylase Inhibitors by Virtual Screening and Fragment-Based Approaches.

As part of our effort to inhibit bacterial fatty acid biosynthesis through the recently validated target biotin carboxylase, we employed a unique combination of two emergent lead discovery strategies. We used both de novo fragment-based drug discovery and virtual screening, which employs 3D shape and electrostatic property similarity searching. We screened a collection of unbiased low-molecular-weight molecules and identified a structurally diverse collection of weak-binding but ligand-efficient fragments as potential building blocks for biotin carboxylase ATP-competitive inhibitors. Through iterative cycles of structure-based drug design relying on successive fragment costructures, we improved the potency of the initial hits by up to 3000-fold while maintaining their ligand-efficiency and desirable physicochemical properties. In one example, hit-expansion efforts resulted in a series of amino-oxazoles with antibacterial activity. These results successfully demonstrate that virtual screening approaches can substantially augment fragment-based screening approaches to identify novel antibacterial agents.

Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

Bacterial acetyl‐CoA carboxylase is a multifunctional biotin‐dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl‐CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half‐reaction, BC catalyzes the ATP‐dependent carboxylation of BCCP. In the second half‐reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl‐CoA to produce malonyl‐CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active‐site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate‐induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half‐sites reactivity where the active sites alternate or “flip‐flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half‐sites reactivity in BC.

Crystal structure of LpxC from Pseudomonas aeruginosa complexed with the potent BB-78485 inhibitor

The cell wall in Gram‐negative bacteria is surrounded by an outer membrane comprised of charged lipopolysaccharide (LPS) molecules that prevent entry of hydrophobic agents into the cell and protect the bacterium from many antibiotics. The hydrophobic anchor of LPS is lipid A, the biosynthesis of which is essential for bacterial growth and viability. UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine deacetylase (LpxC) is an essential zinc‐dependant enzyme that catalyzes the conversion of UDP‐3‐O‐(R‐3‐hydroxymyristoyl)‐N‐acetylglucosamine to UDP‐3‐O‐(R‐3‐hydroxymyristoyl)glucosamine and acetate in the biosynthesis of lipid A, and for this reason, LpxC is an attractive target for antibacterial drug discovery. Here we disclose a 1.9 Å resolution crystal structure of LpxC from Pseudomonas aeruginosa (paLpxC) in a complex with the potent BB‐78485 inhibitor. To our knowledge, this is the first crystal structure of LpxC with a small‐molecule inhibitor that shows antibacterial activity against a wide range of Gram‐negative pathogens. Accordingly, this structure can provide important information for lead optimization and rational design of the effective small‐molecule LpxC inhibitors for successful treatment of Gram‐negative infections.

Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

N‐Acetylglucosamine‐1‐phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine‐diphospho‐N‐acetylglucosamine (UDP‐GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram‐negative and Gram‐positive bacteria. Here we disclose a 1.9 Å resolution crystal structure of a synthetic small‐molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high‐throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC50 ∼ 18 μM in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc‐1‐P substrate‐binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure‐guided design of a new class of mechanism‐based inhibitors of GlmU.

Characterization of substrate binding and catalysis in the potential antibacterial target N-acetylglucosamine-1-phosphate uridyltransferase (GlmU)

N‐Acetylglucosamine‐1‐phosphate uridyltransferase (GlmU) catalyzes the first step in peptidoglycan biosynthesis in both Gram‐positive and Gram‐negative bacteria. The products of the GlmU reaction are essential for bacterial survival, making this enzyme an attractive target for antibiotic drug discovery. A series of Haemophilus influenzae GlmU (hiGlmU) structures were determined by X‐ray crystallography in order to provide structural and functional insights into GlmU activity and inhibition. The information derived from these structures was combined with biochemical characterization of the K25A, Q76A, D105A, Y103A, V223A, and E224A hiGlmU mutants in order to map these active‐site residues to catalytic activity of the enzyme and refine the mechanistic model of the GlmU uridyltransferase reaction. These studies suggest that GlmU activity follows a sequential substrate‐binding order that begins with UTP binding noncovalently to the GlmU enzyme. The uridyltransferase active site then remains in an open apo‐like conformation until N‐acetylglucosamine‐1‐phosphate (GlcNAc‐1‐P) binds and induces a conformational change at the GlcNAc‐binding subsite. Following the binding of GlcNAc‐1‐P to the UTP‐charged uridyltransferase active site, the non‐esterified oxygen of GlcNAc‐1‐P performs a nucleophilic attack on the α‐phosphate group of UTP. The new data strongly suggest that the mechanism of phosphotransfer in the uridyltransferase reaction in GlmU is primarily through an associative mechanism with a pentavalent phosphate intermediate and an inversion of stereochemistry. Finally, the structural and biochemical characterization of the uridyltransferase active site and catalytic mechanism described herein provides a basis for the structure‐guided design of novel antibacterial agents targeting GlmU activity.

Structure based design of an in vivo active hydroxamic acid inhibitor of P. aeruginosa LpxC

Lipid A is an essential component of the Gram negative outer membrane, which protects the bacterium from attack of many antibiotics. The Lipid A biosynthesis pathway is essential for Gram negative bacterial growth and is unique to these bacteria. The first committed step in Lipid A biosynthesis is catalysis by LpxC, a zinc dependent deacetylase. We show the design of an LpxC inhibitor utilizing a robust model which directed efficient design of picomolar inhibitors. Analysis of physiochemical properties drove design to focus on an optimal lipophilicity profile. Further structure based design took advantage of a conserved water network over the active site, and with the optimal lipophilicity profile, led to an improved LpxC inhibitor with in vivo activity against wild type Pseudomonas aeruginosa.

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild‐type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.

Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

Discovery of potent, selective, bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model, Part I: Transformation of selective pyrazolodiazepinone phosphodiesterase 4 (PDE4) inhibitors into selective PDE2 inhibitors.

We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of PDE4 inhibitors, while simultaneously minimizing PDE4 activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like mode in contrast to the cAMP-like binding mode found in PDE4. Structure activity relationship studies coupled with an inhibitor bound crystal structure in the active site of the catalytic domain of PDE2 identified structural features required to minimize PDE4 inhibition while simultaneously maximizing PDE2 inhibition.

Discovery of potent selective bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model. Part II: optimization studies and demonstration of in vivo efficacy.

Selective phosphodiesterase 2 (PDE2) inhibitors are shown to have efficacy in a rat model of osteoarthritis (OA) pain. We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of phosphodiesterase 4 (PDE4) inhibitors, while minimizing PDE4 inhibitory activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like binding mode orthogonal to the cAMP-like binding mode found in PDE4. Extensive structure activity relationship studies ultimately led to identification of pyrazolodiazepinone, 22, which was >1000-fold selective for PDE2 over recombinant, full length PDEs 1B, 3A, 3B, 4A, 4B, 4C, 7A, 7B, 8A, 8B, 9, 10 and 11. Compound 22 also retained excellent PDE2 selectivity (241-fold to 419-fold) over the remaining recombinant, full length PDEs, 1A, 4D, 5, and 6. Compound 22 exhibited good pharmacokinetic properties and excellent oral bioavailability (F=78%, rat). In an in vivo rat model of OA pain, compound 22 had significant analgesic activity 1 and 3h after a single, 10 mg/kg, subcutaneous dose.