Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.

N-Hydroxyformamide LpxC inhibitors, their in vivo efficacy in a mouse Escherichia coli infection model, and their safety in a rat hemodynamic assay.

UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC), the zinc metalloenzyme catalyzing the first committed step of lipid A biosynthesis in Gram-negative bacteria, has been a target for antibacterial drug discovery for many years. All inhibitor chemotypes reaching an advanced preclinical stage and clinical phase 1 have contained terminal hydroxamic acid, and none have been successfully advanced due, in part, to safety concerns, including hemodynamic effects. We hypothesized that the safety of LpxC inhibitors could be improved by replacing the terminal hydroxamic acid with a different zinc-binding group. After choosing an N-hydroxyformamide zinc-binding group, we investigated the structure-activity relationship of each part of the inhibitor scaffold with respect to Pseudomonas aeruginosa and Escherichia coli LpxC binding affinity, in vitro antibacterial potency and pharmacological properties. We identified a novel, potency-enhancing hydrophobic binding interaction for an LpxC inhibitor. We demonstrated in vivo efficacy of one compound in a neutropenic mouse E. coli infection model. Another compound was tested in a rat hemodynamic assay and was found to have a hypotensive effect. This result demonstrated that replacing the terminal hydroxamic acid with a different zinc-binding group was insufficient to avoid this previously recognized safety issue with LpxC inhibitors.

5-Carboxytetramethylrhodamine-Ampicillin Fluorescence Anisotropy-Based Assay of Escherichia coli Penicillin-Binding Protein 2 Transpeptidase Inhibition.

The high-molecular mass penicillin-binding proteins (PBPs) are the essential targets of the ß-lactam classes of antibacterial drugs. In the Gram-negative pathogen Escherichia coli, these include PBP1a, PBP1b, PBP2, and PBP3. Techniques that enable facile measurement of the potency of inhibition of these targets are valuable for understanding structure-activity relationships in programs aimed at discovering new antibiotics to combat drug-resistant infections. Continuous fluorescence anisotropy-based assays for inhibition of soluble constructs of PBP1a, PBP2, and PBP3 from the serious Gram-negative bacterial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii and PBP3 from E. coli using the fluorescent phenoxypenicillin analogue BOCILLIN FL have been described previously, but this technique was not useful for PBP2 from E. coli due to a lack of change in fluorescence anisotropy or intensity upon reaction. Here, we report that a fluorescent analogue of ampicillin, 5-carboxytetramethylrhodamine-ampicillin (5-TAMRA-ampicillin), was useful as the indicator in a continuous fluorescence anisotropy-based kinetic assay for inhibition of a soluble construct of PBP2 from E. coli. The assay enables measurement of the bimolecular rate constant for inhibition kinact /Ki. This measurement was made for representative drugs from four classes of ß-lactams and for the diazabicyclooctenone ETX2514. 5-TAMRA-ampicillin was also useful in a fluorescence anisotropy-based assay for P. aeruginosa PBP2 and in fluorescence intensity-based assays with PBP1a and PBP3 from P. aeruginosa and A. baumannii and PBP3 from E. coli.

An amine protecting group deprotectable under nearly neutral oxidative conditions.

The 1,3-dithiane-based dM-Dmoc group was studied for the protection of amino groups. Protection was achieved under mild conditions for aliphatic amines, and under highly reactive conditions for the less reactive arylamines. Moderate to excellent yields were obtained. Deprotection was performed by oxidation followed by treating with a weak base. The yields were good to excellent. The new amino protecting group offers a different dimension of orthogonality in reference to the commonly used amino protecting groups in terms of deprotection conditions. It is expected to allow a collection of transformations to be carried out on the protected substrates that are unattainable using any known protecting groups.

Whole-Cell-Based Assay To Evaluate Structure Permeation Relationships for Carbapenem Passage through the Pseudomonas aeruginosa Porin OprD.

The global emergence of antibiotic resistance, especially in Gram-negative bacteria, is an urgent threat to public health. Discovery of novel classes of antibiotics with activity against these pathogens has been impeded by a fundamental lack of understanding of the molecular drivers underlying small molecule uptake. Although it is well-known that outer membrane porins represent the main route of entry for small, hydrophilic molecules across the Gram-negative cell envelope, the structure-permeation relationship for porin passage has yet to be defined. To address this knowledge gap, we developed a sensitive and specific whole-cell approach in Escherichia coli called titrable outer membrane permeability assay system (TOMAS). We used TOMAS to characterize the structure porin-permeation relationships of a set of novel carbapenem analogues through the Pseudomonas aeruginosa porin OprD. Our results show that small structural modifications, especially the number and nature of charges and their position, have dramatic effects on the ability of these molecules to permeate cells through OprD. This is the first demonstration of a defined relationship between specific molecular changes in a substrate and permeation through an isolated porin. Understanding the molecular mechanisms that impact antibiotic transit through porins should provide valuable insights to antibacterial medicinal chemistry and may ultimately allow for the rational design of porin-mediated uptake of small molecules into Gram-negative bacteria.

Modulating the strength of hydrogen bond acceptors to achieve low Caco2 efflux for oral bioavailability of PARP inhibitors blocking centrosome clustering.

During the lead generation and optimization of PARP inhibitors blocking centrosome clustering, it was discovered that increasing hydrogen bond acceptor (HBA) strength improved cellular potency but led to elevated Caco2 and MDR1 efflux and thus poor oral bioavailability. Conversely, compounds with lower efflux had reduced potency. The project team was able to improve the bioavailability by reducing efflux through systematic modifications to the strength of the HBA by changing the electronic properties of neighboring groups, whilst maintaining sufficient acceptor strength for potency. Additionally, it was observed that enantiomers with different potency showed similar efflux, which is consistent with the promiscuity of efflux transporters. Eventually, a balance between potency and low efflux was achieved for a set of lead compounds with good bioavailability which allowed the project to progress towards establishing in vivo pharmacokinetic/pharmacodynamic relationships.

Discovery of AZ0108, an orally bioavailable phthalazinone PARP inhibitor that blocks centrosome clustering.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

SAR and Structural Analysis of Siderophore-Conjugated Monocarbam Inhibitors of Pseudomonas aeruginosa PBP3.

A main challenge in the development of new agents for the treatment of Pseudomonas aeruginosa infections is the identification of chemotypes that efficiently penetrate the cell envelope and are not susceptible to established resistance mechanisms. Siderophore-conjugated monocarbams are attractive because of their ability to hijack the bacteria's iron uptake machinery for transport into the periplasm and their inherent stability to metallo-ß-lactamases. Through development of the SAR we identified a number of modifications to the scaffold that afforded active anti-P. aeruginosa agents with good physicochemical properties. Through crystallographic efforts we gained a better understanding into how these compounds bind to the target penicillin binding protein PBP3 and factors to consider for future design.

Toward the rational design of carbapenem uptake in Pseudomonas aeruginosa.

Understanding how compound penetration occurs across the complex cell walls of Gram-negative bacteria is one of the greatest challenges in discovering new drugs to treat the infections they cause. A combination of next-generation transposon sequencing, computational metadynamics simulations (CMDS), and medicinal chemistry was used to define genetic and structural elements involved in facilitated carbapenem entry into Pseudomonas aeruginosa. Here we show for the first time that these compounds are taken up not only by the major outer membrane channel OccD1 (also called OprD or PA0958) but also by a closely related channel OccD3 (OpdP or PA4501). Transport-mediating molecular interactions predicted by CMDS for these channels were first confirmed genetically, then used to guide the design of carbapenem analogs with altered uptake properties. These results bring us closer to the rational design of channel transmissibility and may ultimately lead to improved permeability of compounds across bacterial outer membranes.

Discovery of efficacious Pseudomonas aeruginosa-targeted siderophore-conjugated monocarbams by application of a semi-mechanistic pharmacokinetic/pharmacodynamic model.

To identify new agents for the treatment of multi-drug-resistant Pseudomonas aeruginosa, we focused on siderophore-conjugated monocarbams. This class of monocyclic ß-lactams are stable to metallo-ß-lactamases and have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker, siderophore, and oxime portion of the molecules were examined. Through these efforts, a series of pyrrolidinone-based monocarbams with good P. aeruginosa cellular activity (P. aeruginosa MIC90 = 2 µg/mL), free fraction levels (>20% free), and hydrolytic stability (t1/2 ≥ 100 h) were identified. To differentiate the lead compounds and enable prioritization for in vivo studies, we applied a semi-mechanistic pharmacokinetic/pharmacodynamic model to enable prediction of in vivo efficacy from in vitro data.

Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening.

A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series.

Discovery of spirofused piperazine and diazepane amides as selective histamine-3 antagonists with in vivo efficacy in a mouse model of cognition.

A new series of potent and selective histamine-3 receptor (H3R) antagonists was identified on the basis of an azaspiro[2.5]octane carboxamide scaffold. Many scaffold modifications were largely tolerated, resulting in nanomolar-potent compounds in the H3R functional assay. Exemplar compound 6s demonstrated a selective profile against a panel of 144 secondary pharmacological receptors, with activity at only σ2 (62% at 10 µM). Compound 6s demonstrated free-plasma exposures above the IC50 (∼50×) with a brain-to-plasma ratio of ∼3 following intravenous dosing in mice. At three doses tested in the mouse novel object recognition model (1, 3, and 10 mg/kg s.c.), 6s demonstrated a statistically significant response compared with the control group. This series represents a new scaffold of H3 receptor antagonists that demonstrates in vivo exposure and efficacy in an animal model of cognition.

2,6-Disubstituted pyrazines and related analogs as NR2B site antagonists of the NMDA receptor with anti-depressant activity.

Herein we describe the discovery of compounds that are competitive antagonists of the CP101-606 binding site within the NR2B subtype of the NMDA receptor. The compounds identified do not possess phenolic functional groups such as those in ifenprodil and related analogs. Initial identification of hits in this series focused on a basic, secondary amine side chain which led to good potency, but also presented a hERG liability. Further modifications led to examples of non-basic replacements which demonstrated much less liability in this regard. Finally, one compound in the series, 6a, was tested in the mouse forced swim depression assay and found to show activity (s.c. 60 mg/kg).

Addiction liability of pharmacotherapeutic interventions in obesity.

Obesity and substance use disorders are rapidly growing problems throughout the world. Of the current mainstay therapies of diet, exercise, behavioral modification, surgery, and medications, drugs have the greatest risk for abuse and dependence. As each of these disorders share similar underpinnings mediated by the dopaminergic brain reward pathways, clinicians must seriously consider the safety of both the patient's physical and mental health when prescribing treatments. Specifically, balance and awareness of the factors involved in the variable abuse potentials of these prescribed medications is paramount. A cursory review of weight loss medications commonly used is performed with attention to FDA status, mechanism of action, and abuse potential. Concurrent strategies to minimize risk such as drug screening, ruling out doctor shopping, temporal considerations, monitoring for signs and symptoms of abuse and/or dependency, and a safety-tiered prescribing approach is also discussed in order to optimize best treatment practice. As the understanding of these disorders progresses along with the evolution of agreed nomenclature and awareness of compulsive behavioral disorders in general, greater safety and more appropriate interventions may be achieved. Further areas of research will help to elucidate nuances of the coocurrance and treatment of these disorders and perhaps guide drug research and development in the area of drug treatments of obesity.

[¹²5I]YP20: a novel radioligand specific for the extracellular domain of the CRF1 receptor.

The peptide corticotropin-releasing factor (CRF) binds to the CRF1 receptor via a two-domain mechanism such that the extracellular domain (ECD) of the receptor captures the CRF's C-terminus to facilitate the binding of CRF's N-terminus to the juxta-membrane or "J"-site. Known small molecule antagonists bind to the J-site while known CRF1 receptor peptide radioligands bind to both sites. We report here the in vitro binding properties of the first radioligand that binds exclusively to the ECD of the CRF1 receptor. This ligand, which we named [¹²5I]Yamada peptide 20 ([¹²5I]YP20), is a radiolabeled analog of a synthetic peptide first reported by Yamada et al. (2004). We confirmed its high affinity for the [¹²5I]CRF binding site on the hCRF1 receptor and also found it to potently antagonize CRF-stimulated cAMP production in hCRF1-CHO cells. Under optimized conditions, 20 pM [¹²5I]YP20 reproducibly bound to hCRF1-CHO membranes with a pharmacology consistent with binding specific to the ECD of the CRF1 receptor. Saturation binding studies revealed the presence of a high affinity site with an estimated K(d) of ≈0.9 nM. The kinetic association of 20 pM [¹²5I]YP20 binding best fit to a rapid component (t(1/2)=0.69 min) and a sluggish component (t(1/2)=42 min). [¹²5I]YP20's specific binding was rapidly reversible with dissociation kinetics also best described by two phases (t(1/2)=0.92 min and t(1/2)=11.7 min). While [¹²5I]YP20's binding kinetics are complex, its high affinity and pharmacological specificity indicate that it is an excellent radioligand for probing the ECD site of the CRF1 receptor.

Application of fragment-based lead generation to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency.

Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.

Experimental evaluation of an elastic foundation model to predict contact pressures in knee replacements.

Computational wear prediction is an attractive concept for evaluating new total knee replacement designs prior to physical testing and implementation. An important hurdle to such technology is the lack of in vivo contact pressure predictions. To address this issue, this study evaluates a computationally efficient simulation approach that combines the advantages of rigid and deformable body modeling. The hybrid method uses rigid body dynamics to predict body positions and orientations and elastic foundation theory to predict contact pressures between general three-dimensional surfaces. To evaluate the method, we performed static pressure experiments with a commercial knee implant in neutral alignment using flexion angles of 0, 30, 60, and 90 degrees and loads of 750, 1500, 2250, and 3000N. Using manufacturer CAD geometry for the same implant, an elastic foundation model with linear or nonlinear polyethylene material properties was implemented within a commercial multibody dynamics software program. The model's ability to predict experimental peak and average contact pressures simultaneously was evaluated by performing dynamic simulations to find the static configuration. Both the linear and nonlinear material models predicted the average contact pressure data well, while only the linear material model could simultaneously predict the trends in the peak contact pressure data. This novel modeling approach is sufficiently fast and accurate to be used in design sensitivity and optimization studies of knee implant mechanics and ultimately wear.

Linear non-competitive inhibition of solubilized human gamma-secretase by pepstatin A methylester, L685458, sulfonamides, and benzodiazepines.

Cerebral deposition of amyloid beta-protein (A beta) is believed to play a key role in the pathogenesis of Alzheimer's disease. Because A beta is produced from the processing of amyloid beta-protein precursor (APP) by beta- and gamma-secretases, these enzymes are considered important therapeutic targets for identification of drugs to treat Alzheimer's disease. Unlike beta-secretase, which is a monomeric aspartyl protease, gamma-secretase activity resides as part of a membrane-bound, high molecular weight, macromolecular complex. Pepstatin and L685458 are among several structural classes of gamma-secretase inhibitors identified so far. These compounds possess a hydroxyethylene dipeptide isostere of aspartyl protease transition state analogs, suggesting gamma-secretase may be an aspartyl protease. However, the mechanism of inhibition of gamma-secretase by pepstatin and L685458 has not been elucidated. In this study, we report that pepstatin A methylester and L685458 unexpectedly displayed linear non-competitive inhibition of gamma-secretase. Sulfonamides and benzodiazepines, which do not resemble transition state analogs of aspartyl proteases, also displayed potent, non-competitive inhibition of gamma-secretase. Models to rationalize how transition state analogs inhibit their targets by non-competitive inhibition are discussed.