A covalent BTK ternary complex compatible with targeted protein degradation.

Targeted protein degradation using heterobifunctional chimeras holds the potential to expand target space and grow the druggable proteome. Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Limiting this potential, however, is the remaining need to develop a ligand for the target of interest. While a number of challenging proteins have been successfully targeted by covalent ligands, unless this modification affects form or function, it may lack the ability to drive a biological response. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields. In this work, we employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in targeted protein degradation using Bruton's tyrosine kinase. Our results reveal that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.

Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes.

Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1 H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1ß1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of ß1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1ß1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Optimization of Metabolic and Renal Clearance in a Series of Indole Acid Direct Activators of 5'-Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

Biophysical Interactions of Direct AMPK Activators.

Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.

Discovery of Fragment-Derived Small Molecules for in Vivo Inhibition of Ketohexokinase (KHK).

Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.

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.

Structural basis for AMPK activation: natural and synthetic ligands regulate kinase activity from opposite poles by different molecular mechanisms.

AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive. Challenges result, in part, from an incomplete understanding of the structure and function of full-length heterotrimeric complexes. In this work, we provide the full-length structure of the widely expressed α1ß1γ1 isoform of mammalian AMPK, along with detailed kinetic and biophysical characterization. We characterize binding of the broadly studied synthetic activator A769662 and its analogs. Our studies follow on the heels of the recent disclosure of the α2ß1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.

Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro.

Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 µM) compared with wild-type EGF-A (KD = 1.2 µM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.

Spirocyclic sulfamides as ß-secretase 1 (BACE-1) inhibitors for the treatment of Alzheimer's disease: utilization of structure based drug design, WaterMap, and CNS penetration studies to identify centrally efficacious inhibitors.

ß-Secretase 1 (BACE-1) is an attractive therapeutic target for the treatment and prevention of Alzheimer's disease (AD). Herein, we describe the discovery of a novel class of BACE-1 inhibitors represented by sulfamide 14g, using a medicinal chemistry strategy to optimize central nervous system (CNS) penetration by minimizing hydrogen bond donors (HBDs) and reducing P-glycoprotein (P-gp) mediated efflux. We have also taken advantage of the combination of structure based drug design (SBDD) to guide the optimization of the sulfamide analogues and the in silico tool WaterMap to explain the observed SAR. Compound 14g is a potent inhibitor of BACE-1 with excellent permeability and a moderate P-gp liability. Administration of 14g to mice produced a significant, dose-dependent reduction in central Aß(X-40) levels at a free drug exposure equivalent to the whole cell IC(50) (100 nM). Furthermore, studies of the P-gp knockout mouse provided evidence that efflux transporters affected the amount of Aß lowering versus that observed in wild-type (WT) mouse at an equivalent dose.

Discovery and optimization of a novel spiropyrrolidine inhibitor of ß-secretase (BACE1) through fragment-based drug design.

The aspartyl protease ß-secretase, or BACE, has been demonstrated to be a key factor in the proteolytic formation of Aß-peptide, a major component of plaques in the brains of Alzheimer's disease (AD) patients, and inhibition of this enzyme has emerged as a major strategy for pharmacologic intervention in AD. An X-ray-based fragment screen of Pfizer's proprietary fragment collection has resulted in the identification of a novel BACE binder featuring spiropyrrolidine framework. Although exhibiting only weak inhibitory activity against the BACE enzyme, the small compound was verified by biophysical and NMR-based methods as a bona fide BACE inhibitor. Subsequent optimization of the lead compound, relying heavily on structure-based drug design and computational prediction of physiochemical properties, resulted in a nearly 1000-fold improvement in potency while maintaining ligand efficiency and properties predictive of good permeability and low P-gp liability.

Discovery, SAR, and pharmacokinetics of a novel 3-hydroxyquinolin-2(1H)-one series of potent D-amino acid oxidase (DAAO) inhibitors.

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum D-serine.

Cyclization of N-terminal S-carbamoylmethylcysteine causing loss of 17 Da from peptides and extra peaks in peptide maps.

Enzymatic digests of proteins S-alkylated with iodoacetamide may contain peptides with N-terminal S-carbamoylmethylcysteine. These can be partly converted to a form with 17 Da lower mass and increased HPLC retention. Proof by synthesis supported by MS/MS and NMR spectroscopy was used to show that N-terminal S-carbamoylmethyl-L-cysteine can cyclize, losing NH3 to form an N-terminal residue of (R)-5-oxoperhydro-1,4-thiazine-3-carboxylic acid. The abbreviation Otc is proposed for the (R)-5-oxoperhydro-1,4-thiazine-3-carbonyl residue. The rate of cyclization is significant in 0.1 M NH4HCO3 at 37 degrees C, with the half-life of the acyclic form being 10-12 h for several peptides tested. This is similar to the rate at which N-terminal pyroglutamate forms from N-terminal glutamine.