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We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment., The 3CL protease of SARS-CoV-2 is inhibited by PF-00835231 in vitro. Here, the authors show that the prodrug PF-07304814 has broad spectrum activity, inhibiting SARS-CoV and SARS-CoV-2 in mice and its ADME and safety profile support clinical development.

A major challenge in the development of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors for the treatment of Alzheimer's disease is the alignment of potency, drug-like properties, and selectivity over related aspartyl proteases such as Cathepsin D (CatD) and BACE2. The potential liabilities of inhibiting BACE2 chronically have only recently begun to emerge as BACE2 impacts the processing of the premelanosome protein (PMEL17) and disrupts melanosome morphology resulting in a depigmentation phenotype. Herein, we describe the identification of clinical candidate PF-06751979 (64), which displays excellent brain penetration, potent in vivo efficacy, and broad selectivity over related aspartyl proteases including BACE2. Chronic dosing of 64 for up to 9 months in dog did not reveal any observation of hair coat color (pigmentation) changes and suggests a key differentiator over current BACE1 inhibitors that are nonselective against BACE2 in later stage clinical development.

We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI) MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision and accuracy; subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro and in vivo adsorption, distribution, metabolism, elimination, pharmacokinetic (PK) and biomarker analysis, and HT parallel medicinal chemistry.One Sentence SummaryADE-OPI-MS is a transformational analytical platform that increases mass spectrometry utility via sub-second speed and non-contact sampling.

Apolipoprotein E (apoE) is a 34 kDa key lipid transport protein in both the brain and the periphery. In the brain, it is produced predominantly by astrocytes and has been shown to play an important role in promoting neurite outgrowth and synaptogenesis, clearing amyloid β42, promoting cerebrovascular integrity and in neuroimmune modulation. Given the critical role of apoE in the brain, it is important to understand its regulation in the CNS. To that end, utilizing a phenotypic screen, we identified novel chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe and carried out quantitative chemical proteomics to identify liver x receptor β (LXRβ) as the target. Binding of the ligand stabilized LXRβ, as shown by CETSA. Our findings demonstrated that the lead chemical matter bound directly to LXRβ, and highlight the power of chemical proteomics to identify the target of a phenotypic screening hit.

A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer's disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug-drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.

Summary Utilizing a phenotypic screen, we identified chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe based on a pyrrolidine lead compound and carried out probe-based quantitative chemical proteomics in human astrocytoma CCF-STTG1 cells to identify liver x receptor β (LXRβ) as the target. Binding of the small molecule ligand stabilized LXRβ, as shown by cellular thermal shift assay (CETSA). In addition, we identified a probe-modified peptide by mass spectrometry and proposed a model where the photoaffinity probe is bound in the ligand-binding pocket of LXRβ. Taken together, our findings demonstrated that the lead chemical matter bound directly to LXRβ, and our results highlight the power of chemical proteomic approaches to identify the target of a phenotypic screening hit. Additionally, the LXR photoaffinity probe and lead compound described herein may serve as valuable tools to further evaluate the LXR pathway.

Alzheimer’s disease (AD) is characterized by accumulation of β-amyloid (Aβ) plaques and neurofibrillary tau tangles in the brain. β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a key role in the generation of Aβ fragments via extracellular cleavage of the amyloid precursor protein (APP). We became interested in developing a BACE1 PET ligand to facilitate clinical assessment of BACE1 inhibitors and explore its potential in the profiling and selection of patients for AD trials. Using a set of PET ligand design parameters, compound 3 (PF-06684511) was rapidly identified as a lead with favorable in vitro attributes and structural handles for PET radiolabeling. Further evaluation in an LC-MS/MS “cold tracer” study in rodents revealed high specific binding to BACE1 in brain. Upon radiolabeling, [18F]3 demonstrated favorable brain uptake and high in vivo specificity in nonhuman primate (NHP), suggesting its potential for imaging BACE1 in humans.

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. 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 Aβ(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 Aβ lowering versus that observed in wild-type (WT) mouse at an equivalent dose.

Binding to sigma sites in subcellular fractions of brain and in crude homogenates from peripheral tissues of the guinea pig was characterized with the [3H]ligands (+)pentazocine and di(2-tolyl)guanidine (DTG). The inhibitory effects of representative sigma compounds and cytochrome P450 inhibitors were evaluated in guinea pig tissues, and the effects of cytochrome P450 induction on sigma binding in the rat were investigated. For both ligands, the majority of sites were localized to the microsomal fractions. The KD values for [3H](+)pentazocine- or [3H]DTG-labeled sigma sites in guinea pig liver and testes were 2-fold lower than those in brain and heart. The number of sites labeled by [3H](+)pentazocine varied, with an order of livertestesbrainheart. In contrast, the Bmax values for [3H]DTG-defined sigma sites were greatest in testes, followed by liver, brain and heart. The rank order of potency for representative sigma and P450 compounds was similar in brain, liver and testes for both [3H]ligands, and the potency of selective compounds to displace sigma binding in guinea pig liver failed to correlate with their abilities to inhibit cytochrome P450IID1 activity in human liver. Following induction of cytochrome P450IIB1 with phenobarbital or cytochrome P450IA1 with beta-naphthoflavone, neither the affinity nor the number of sigma sites was altered in rat brain or liver. These results suggest that sigma sites in the periphery are similar to those in the brain, and that the sigma binding site is not identical with cytochrome P450IIB1, P450IA1 or P450IID1.

The synthesis and structure–activity relationships for a novel series of 6-amino-4-(pyrimidin-4-yl)pyridones derived from a high throughput screening hit are discussed. Optimization of lead matter afforded compounds with good potency, selectivity and central nervous system (CNS) exposure.