Intramolecular Ring-Opening Decomposition of Aryl Azetidines.

The ring strain present in azetidines can lead to undesired stability issues. Herein, we described a series of N-substituted azetidines which undergo an acid-mediated intramolecular ring-opening decomposition via nucleophilic attack of a pendant amide group. Studies were conducted to understand the decomposition mechanism enabling the design of stable analogues.

PF-07059013: A Noncovalent Modulator of Hemoglobin for Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (β6 Glu → Val) on the β-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke.

NMR spectroscopy: the swiss army knife of drug discovery.

Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool within drug discovery over the last two decades. While traditionally being used by medicinal chemists for small molecule structure elucidation, it can also be a valuable tool for the identification of small molecules that bind to drug targets, for the characterization of target-ligand interactions and for hit-to-lead optimization. Here, we describe how NMR spectroscopy is integrated into the Pfizer drug discovery pipeline and how we utilize this approach to identify and validate initial hits and generate leads.

Aminopyrazole Carboxamide Bruton's Tyrosine Kinase Inhibitors. Irreversible to Reversible Covalent Reactive Group Tuning.

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies.

An Intracellular Allosteric Modulator Binding Pocket in SK2 Ion Channels Is Shared by Multiple Chemotypes.

Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes.

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.

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.

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.

Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

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.

Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide.

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions.

Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators.

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ1, α1β2γ1, α1β2γ3, α2β1γ1, α2β2γ1 and α2β2γ3 using known activators, A769662 and AMP.

Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency.

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.

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.

Spirocyclic sulfamides as β-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.

β-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.

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

The aspartyl protease β-secretase, or BACE, has been demonstrated to be a key factor in the proteolytic formation of Aβ-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.

Glycomimetic ligands for the human asialoglycoprotein receptor.

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions.

Design of a multi-purpose fragment screening library using molecular complexity and orthogonal diversity metrics.

Fragment Based Drug Discovery (FBDD) continues to advance as an efficient and alternative screening paradigm for the identification and optimization of novel chemical matter. To enable FBDD across a wide range of pharmaceutical targets, a fragment screening library is required to be chemically diverse and synthetically expandable to enable critical decision making for chemical follow-up and assessing new target druggability. In this manuscript, the Pfizer fragment library design strategy which utilized multiple and orthogonal metrics to incorporate structure, pharmacophore and pharmacological space diversity is described.

Molecular features crucial to the activity of pyrimidine benzamide-based thrombopoietin receptor agonists.

The identification of small molecule modulators of biological processes mediated via protein-protein interactions has generally proved to be a challenging endeavor. In the case of the thrombopoietin receptor (TPOr), however, a number of small molecule types have been reported to display biological activity similar to that of the agonist protein TPO. Through a detailed analysis of structure-activity relationships, X-ray crystal structures, NMR coupling constants, nuclear Overhauser effects, and computational data, we have determined the agonism-inducing conformation of one series of small molecule TPOr agonists.

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.

Synthesis of macrocyclic, potential protease inhibitors using a generic scaffold.

A generic macrocyclic peptide structure 2 was designed as a potential inhibitor of a range of proteinases, by using as a basis for the design the known structures of a series of enzyme-inhibitor complexes. The macrocyclic nature of the target 2 was chosen so as to reduce the entropic advantage in the hydrolytic enzymatic step, and thereby to inhibit the function of the enzyme. The nature of the linking group was identified as a benzoxazole by molecular modeling, so as to preserve the recognized conformation of the peptide chain.