Nicole Caspers

Imidazo(1,5-a)quinoxalines as irreversible BTK inhibitors for the treatment of rheumatoid arthritis

Imidazo[1,5-a]quinoxalines were synthesized that function as irreversible Brutons tyrosine kinase (BTK) inhibitors. The syntheses and SAR of this series of compounds are presented as well as the X-ray crystal structure of the lead compound 36 in complex with a gate-keeper variant of ITK enzyme. The lead compound showed good in vivo efficacy in preclinical RA models.

Structural and Inhibition Analysis Reveals the Mechanism of Selectivity of a Series of Aggrecanase Inhibitors

Several inhibitors of a series of cis-1(S)2(R)-amino-2-indanol-based compounds were reported to be selective for the aggrecanases, ADAMTS-4 and -5 over other metalloproteases. To understand the nature of this selectivity for aggrecanases, the inhibitors, along with the broad spectrum metalloprotease inhibitor marimastat, were independently bound to the catalytic domain of ADAMTS-5, and the corresponding crystal structures were determined. By comparing the structures, it was determined that the specificity of the relative inhibitors for ADAMTS-5 was not driven by a specific interaction, such as zinc chelation, hydrogen bonding, or charge interactions, but rather by subtle and indirect factors, such as water bridging, ring rigidity, pocket size, and shape, as well as protein conformation flexibility.

2-(6-Phenyl-1H-indazol-3-yl)-1H-benzo[d]imidazoles: Design and synthesis of a potent and isoform selective PKC-[zeta] inhibitor

The inhibition of PKC-zeta has been proposed to be a potential drug target for immune and inflammatory diseases. A series of 2-(6-phenyl-1H indazol-3-yl)-1H-benzo[d]imidazoles with initial high crossover to CDK-2 has been optimized to afford potent and selective inhibitors of protein kinase c-zeta (PKC-zeta). The determination of the crystal structures of key inhibitor:CDK-2 complexes informed the design and analysis of the series. The most selective and potent analog was identified by variation of the aryl substituent at the 6-position of the indazole template to give a 4-NH(2) derivative. The analog displays good selectivity over other PKC isoforms (alpha, betaII, gamma, delta, epsilon, mu, theta, eta and iota/lambda) and CDK-2, however it displays marginal selectivity against a panel of other kinases (37 profiled).

Covalent inhibitors of interleukin-2 inducible T cell kinase (itk) with nanomolar potency in a whole-blood assay.

We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.

Benzothiophene inhibitors of MK2. Part 1: structure-activity relationships, assessments of selectivity and cellular potency

Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.

Discovery and Preclinical Characterization of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577), a Direct Activator of Adenosine Monophosphate-activated Protein Kinase (AMPK), for the Potential Treatment of Diabetic Nephropathy.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

Thermodynamic and Structure Guided Design of Statin Based Inhibitors of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Clinical studies have demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors, are effective at lowering mortality levels associated with cardiovascular disease; however, 2-7% of patients may experience statin-induced myalgia that limits compliance with a treatment regimen. High resolution crystal structures, thermodynamic binding parameters, and biochemical data were used to design statin inhibitors with improved HMGR affinity and therapeutic index relative to statin-induced myalgia. These studies facilitated the identification of imidazole 1 as a potent (IC 50 = 7.9 nM) inhibitor with excellent hepatoselectivity (>1000-fold) and good in vivo efficacy. The binding of 1 to HMGR was found to be enthalpically driven with a Delta H of -17.7 kcal/M. Additionally, a second novel series of bicyclic pyrrole-based inhibitors was identified that induced order in a protein flap of HMGR. Similar ordering was detected in a substrate complex, but has not been reported in previous statin inhibitor complexes with HMGR.

Benzothiophene inhibitors of MK2. Part 2: improvements in kinase selectivity and cell potency.

Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

Siderophore Receptor-Mediated Uptake of Lactivicin Analogues in Gram-Negative Bacteria

Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug-target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from Pseudomonas aeruginosa and characterize the reactivity of lactivicin and β-lactam core structures. Results from drug sensitivity studies with β-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.

Distinctive Attributes of β-Lactam Target Proteins in Acinetobacter baumannii Relevant to Development of New Antibiotics

Multi-drug-resistant forms of the Gram-negative pathogen Acinetobacter baumannii are an emerging threat to human health and further complicate the general problem of treating serious bacterial infections. Meeting this challenge requires an improved understanding of the relationships between the structures of major therapeutic targets in this organism and the activity levels exhibited against it by different antibiotics. Here we report the first crystal structures of A. baumannii penicillin-binding proteins (PBPs) covalently inactivated by four β-lactam antibiotics. We also relate the results to kinetic, biophysical, and computational data. The structure of the class A protein PBP1a was solved in apo form and for its covalent conjugates with benzyl penicillin, imipenem, aztreonam, and the siderophore-conjugated monocarbam MC-1. It included a novel domain genetically spliced into a surface loop of the transpeptidase domain that contains three conserved loops. Also reported here is the first high-resolution structure of the A. baumannii class B enzyme PBP3 in apo form. Comparison of this structure with that of MC-1-derivatized PBP3 of Pseudomonas aeruginosa identified differences between these orthologous proteins in A. baumannii and P. aeruginosa. Thermodynamic analyses indicated that desolvation effects in the PBP3 ligand-binding sites contributed significantly to the thermal stability of the enzyme-antibiotic covalent complexes. Across a significant range of values, they correlated well with results from studies of inactivation kinetics and the protein structures. The structural, biophysical, and computational data help rationalize differences in the functional performance of antibiotics against different protein targets and can be used to guide the design of future agents.