Identification of a fibronectin-binding protein signature associated with idiopathic pulmonary fibrosis.

The extracellular matrix (ECM) is a critical component of tissue where it provides structural and signaling support to cells. Its dysregulation and accumulation lead to fibrosis, a major clinical challenge underlying many diseases that currently has little effective treatment. An understanding of the key molecular initiators of fibrosis would be both diagnostically useful and provide potential targets for therapeutics. The ECM protein fibronectin (FN) is upregulated in fibrotic conditions and other ECM proteins depend on assembly of a FN foundational ECM for their matrix incorporation. We used cell culture and in vivo models to investigate the role of FN in the progression of lung fibrosis. We confirmed that normal human lung fibroblasts (NHLFs) treated with transforming growth factor-beta (TGF-ß) to stimulate fibrotic gene expression significantly increased both FN expression and its assembly into a matrix. We found that levels of alternatively spliced EDA and EDB exons were proportional to the increase in total FN RNA and protein showing that inclusion of these exons is not enhanced by TGF-ß stimulation. RNA-sequencing identified 43 core matrisome genes that were significantly up- or down-regulated by TGF-ß treatment and a Luminex immunoassay demonstrated increased levels of ECM proteins in conditioned medium of TGF-ß-treated NHLFs. Interestingly, among the regulated core matrisome genes, 16 encode known FN-binding proteins and, of these, insulin-like growth factor binding protein 3 (IGFBP3) was most highly up-regulated. To link the NHLF results with in vivo disease, we analyzed lung tissue and bronchoalveolar lavage fluid from bleomycin-treated mice and found dramatically higher levels of FN and the FN-binding proteins IGFBP3, tenascin-C, and type I collagen in fibrotic conditions compared to controls. Altogether, our data identify a set of FN-binding proteins whose upregulation is characteristic of IPF and suggest that FN provides the foundational matrix for deposition of these proteins as fibrosis develops.

Perspectives on phenotypic screening-Screen Design and Assay Technology Special Interest Group.

Here we offer perspectives on phenotypic screening based on a wide-ranging discussion entitled "Phenotypic screening, target ID, and multi-omics: enabling more disease relevance in early discovery?" at the Screen Design and Assay Technology Special Interest Group Meeting at the 2023 SLAS Conference. During the session, the authors shared their own experience from within their respective organizations, followed by an open discussion with the audience. It was recognized that while substantial progress has been made towards translating disease-relevant phenotypic early discovery into clinical success, there remain significant operational and scientific challenges to implementing phenotypic screening efforts, and improving translation of screening hits comes with substantial resource demands and organizational commitment. This Perspective assesses progress, highlights pitfalls, and offers possible solutions to help unlock the therapeutic potential of phenotypic drug discovery. Areas explored comprise screening and hit validation strategy, choice of cellular model, moving beyond 2D cell culture into three dimensions, and leveraging high-dimensional data sets downstream of phenotypic screens.

Is atrial fibrillation in HFpEF a distinct phenotype? Insights from multiparametric MRI and circulating biomarkers.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) and atrial fibrillation (AF) frequently co-exist. There is a limited understanding on whether this coexistence is associated with distinct alterations in myocardial remodelling and mechanics. We aimed to determine if patients with atrial fibrillation (AF) and heart failure with preserved ejection fraction (HFpEF) represent a distinct phenotype. METHODS: In this secondary analysis of adults with HFpEF (NCT03050593), participants were comprehensively phenotyped with stress cardiac MRI, echocardiography and plasma fibroinflammatory biomarkers, and were followed for the composite endpoint (HF hospitalisation or death) at a median of 8.5 years. Those with AF were compared to sinus rhythm (SR) and unsupervised cluster analysis was performed to explore possible phenotypes. RESULTS: 136 subjects were included (SR = 75, AF = 61). The AF group was older (76 ± 8 vs. 70 ± 10 years) with less diabetes (36% vs. 61%) compared to the SR group and had higher left atrial (LA) volumes (61 ± 30 vs. 39 ± 15 mL/m2, p < 0.001), lower LA ejection fraction (EF) (31 ± 15 vs. 51 ± 12%, p < 0.001), worse left ventricular (LV) systolic function (LVEF 63 ± 8 vs. 68 ± 8%, p = 0.002; global longitudinal strain 13.6 ± 2.9 vs. 14.7 ± 2.4%, p = 0.003) but higher LV peak early diastolic strain rates (0.73 ± 0.28 vs. 0.53 ± 0.17 1/s, p < 0.001). The AF group had higher levels of syndecan-1, matrix metalloproteinase-2, proBNP, angiopoietin-2 and pentraxin-3, but lower level of interleukin-8. No difference in clinical outcomes was observed between the groups. Three distinct clusters were identified with the poorest outcomes (Log-rank p = 0.029) in cluster 2 (hypertensive and fibroinflammatory) which had equal representation of SR and AF. CONCLUSIONS: Presence of AF in HFpEF is associated with cardiac structural and functional changes together with altered expression of several fibro-inflammatory biomarkers. Distinct phenotypes exist in HFpEF which may have differing clinical outcomes.

Proteomic Correlates of the Urinary Protein/Creatinine Ratio in Heart Failure With Preserved Ejection Fraction.

Proteinuria is common in heart failure with preserved ejection fraction (HFpEF), but its biologic correlates are poorly understood. We assessed the relation between 49 plasma proteins and the urinary protein/creatinine ratio (UPCR) in 365 participants in the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial. Linear regression and network analysis were used to represent relations between protein biomarkers and UPCR. Higher UPCR was associated with older age, a greater proportion of female gender, smaller prevalence of previous myocardial infarction, and greater prevalence of diabetes, insulin use, smoking, and statin use, in addition to a lower estimated glomerular filtration rate, hematocrit, and diastolic blood pressure. Growth differentiation factor 15 (GDF-15; ß = 0.15, p <0.0001), followed by N-terminal proatrial natriuretic peptide (NT-proANP; ß = 0.774, p <0.0001), adiponectin (ß = 0.0005, p <0.0001), fibroblast growth factor 23 (FGF-23, ß = 0.177; p <0.0001), and soluble tumor necrosis factor receptors I (ß = 0.002, p <0.0001) and II (ß = 0.093, p <0.0001) revealed the strongest associations with UPCR. Network analysis showed that UPCR is linked to various proteins primarily through FGF-23, which, along with GDF-15, indicated node characteristics with strong connectivity, whereas UPCR did not. In a model that included FGF-23 and UPCR, the former was predictive of the risk of death or heart-failure hospital admission (standardized hazard ratio 1.83, 95% confidence interval 1.49 to 2.26, p <0.0001) and/or all-cause death (standardized hazard ratio 1.59, 95% confidence interval 1.22 to 2.07, p = 0.0005), whereas UPCR was not prognostic. Proteinuria in HFpEF exhibits distinct proteomic correlates, primarily through its association with FGF-23, a well-known prognostic marker in HFpEF. However, in contrast to FGF-23, UPCR does not hold independent prognostic value.

Impact of diabetes on remodelling, microvascular function and exercise capacity in aortic stenosis.

OBJECTIVE: To characterise cardiac remodelling, exercise capacity and fibroinflammatory biomarkers in patients with aortic stenosis (AS) with and without diabetes, and assess the impact of diabetes on outcomes. METHODS: Patients with moderate or severe AS with and without diabetes underwent echocardiography, stress cardiovascular magnetic resonance (CMR), cardiopulmonary exercise testing and plasma biomarker analysis. Primary endpoint for survival analysis was a composite of cardiovascular mortality, myocardial infarction, hospitalisation with heart failure, syncope or arrhythmia. Secondary endpoint was all-cause death. RESULTS: Diabetes (n=56) and non-diabetes groups (n=198) were well matched for age, sex, ethnicity, blood pressure and severity of AS. The diabetes group had higher body mass index, lower estimated glomerular filtration rate and higher rates of hypertension, hyperlipidaemia and symptoms of AS. Biventricular volumes and systolic function were similar, but the diabetes group had higher extracellular volume fraction (25.9%±3.1% vs 24.8%±2.4%, p=0.020), lower myocardial perfusion reserve (2.02±0.75 vs 2.34±0.68, p=0.046) and lower percentage predicted peak oxygen consumption (68%±21% vs 77%±17%, p=0.002) compared with the non-diabetes group. Higher levels of renin (log10renin: 3.27±0.59 vs 2.82±0.69 pg/mL, p<0.001) were found in diabetes. Multivariable Cox regression analysis showed diabetes was not associated with cardiovascular outcomes, but was independently associated with all-cause mortality (HR 2.04, 95% CI 1.05 to 4.00; p=0.037). CONCLUSIONS: In patients with moderate-to-severe AS, diabetes is associated with reduced exercise capacity, increased diffuse myocardial fibrosis and microvascular dysfunction, but not cardiovascular events despite a small increase in mortality.

Structure-activity relationship study of central pyridine-derived TYK2 JH2 inhibitors: Optimization of the PK profile through C4' and C6 variations.

Efforts directed at improving potency and preparing structurally different TYK2 JH2 inhibitors from the first generation of compounds such as 1a led to the SAR study of new central pyridyl based analogs 2-4. The current SAR study resulted in the identification of 4h as a potent and selective TYK2 JH2 inhibitor with distinct structural differences from 1a. In this manuscript, the in vitro and in vivo profiles of 4h are described. The hWB IC50 of 4h was shown as 41 nM with 94% bioavailability in the mouse PK study.

Discovery of novel pyridinones as MGAT2 inhibitors for the treatment of metabolic disorders.

Inhibition of monoacylglycerol transferase 2 (MGAT2) has recently emerged as a potential therapeutic strategy for the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic steatohepatitis (NASH). Metabolism studies with our clinical lead (1) suggested variability in in vitro glucuronidation rates in liver microsomes across species, which made projection of human doses challenging. In addition, the observation of deconjugation of the C3-C4 double bond in the dihydropyridinone ring of 1 in solution had the potential to complicate its clinical development. This report describes our lead optimization efforts in a novel pyridinone series, exemplified by compound 33, which successfully addressed both of these potential issues.

Optimization of physicochemical properties of pyrrolidine GPR40 AgoPAMs results in a differentiated profile with improved pharmacokinetics and reduced off-target activities.

GPR40 AgoPAMs are highly effective antidiabetic agents that have a dual mechanism of action, stimulating both glucose-dependent insulin and GLP-1 secretion. The early lipophilic, aromatic pyrrolidine and dihydropyrazole GPR40 AgoPAMs from our laboratory were highly efficacious in lowering plasma glucose levels in rodents but possessed off-target activities and triggered rebound hyperglycemia in rats at high doses. A focus on increasing molecular complexity through saturation and chirality in combination with reducing polarity for the pyrrolidine AgoPAM chemotype resulted in the discovery of compound 46, which shows significantly reduced off-target activities as well as improved aqueous solubility, rapid absorption, and linear PK. In vivo, compound 46 significantly lowers plasma glucose levels in rats during an oral glucose challenge yet does not demonstrate the reactive hyperglycemia effect at high doses that was observed with earlier GPR40 AgoPAMs.

Discovery of Two Novel Antiplatelet Clinical Candidates (BMS-986120 and BMS-986141) That Antagonize Protease-Activated Receptor 4.

Protease-activated receptor 4 (PAR4) is a G-protein coupled receptor that is expressed on human platelets and activated by the coagulation enzyme thrombin. PAR4 plays a key role in blood coagulation, and its importance in pathological thrombosis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of imidazothiadiazole PAR4 antagonists to a first-in-class clinical candidate, BMS-986120 (43), and a backup clinical candidate, BMS-986141 (49). Both compounds demonstrated excellent antithrombotic efficacy and minimal bleeding time prolongation in monkey models relative to the clinically important antiplatelet agent clopidogrel and provide a potential opportunity to improve the standard of care in the treatment of arterial thrombosis.

BMS-813160: A Potent CCR2 and CCR5 Dual Antagonist Selected as a Clinical Candidate.

BMS-813160 (compound 3) was identified as a potent and selective CCR2/5 dual antagonist. Compound 3 displayed good permeability at pH = 7.4 in PAMPA experiments and demonstrated excellent human liver microsome stability. Pharmacokinetic studies established that 3 had excellent oral bioavailability and exhibited low clearance in dog and cyno. Compound 3 was also studied in the mouse thioglycollate-induced peritonitis model, which confirmed its ability to inhibit the migration of inflammatory monocytes and macrophages. As a result of this profile, compound 3 was selected as a clinical candidate.

Discovery of an Oxycyclohexyl Acid Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist BMS-986278 for the Treatment of Pulmonary Fibrotic Diseases.

The oxycyclohexyl acid BMS-986278 (33) is a potent lysophosphatidic acid receptor 1 (LPA1) antagonist, with a human LPA1 Kb of 6.9 nM. The structure-activity relationship (SAR) studies starting from the LPA1 antagonist clinical compound BMS-986020 (1), which culminated in the discovery of 33, are discussed. The detailed in vitro and in vivo preclinical pharmacology profiles of 33, as well as its pharmacokinetics/metabolism profile, are described. On the basis of its in vivo efficacy in rodent chronic lung fibrosis models and excellent overall ADME (absorption, distribution, metabolism, excretion) properties in multiple preclinical species, 33 was advanced into clinical trials, including an ongoing Phase 2 clinical trial in patients with lung fibrosis (NCT04308681).

Plasma biomarkers associated with adverse outcomes in patients with calcific aortic stenosis.

AIMS: Enhanced risk stratification of patients with aortic stenosis (AS) is necessary to identify patients at high risk for adverse outcomes, and may allow for better management of patient subgroups at high risk of myocardial damage. The objective of this study was to identify plasma biomarkers and multimarker profiles associated with adverse outcomes in AS. METHODS AND RESULTS: We studied 708 patients with calcific AS and measured 49 biomarkers using a Luminex platform. We studied the correlation between biomarkers and the risk of (i) death and (ii) death or heart failure-related hospital admission (DHFA). We also utilized machine-learning methods (a tree-based pipeline optimizer platform) to develop multimarker models associated with the risk of death and DHFA. In this cohort with a median follow-up of 2.8 years, multiple biomarkers were significantly predictive of death in analyses adjusted for clinical confounders, including tumour necrosis factor (TNF)-α [hazard ratio (HR) 1.28, P < 0.0001], TNF receptor 1 (TNFRSF1A; HR 1.38, P < 0.0001), fibroblast growth factor (FGF)-23 (HR 1.22, P < 0.0001), N-terminal pro B-type natriuretic peptide (NT-proBNP) (HR 1.58, P < 0.0001), matrix metalloproteinase-7 (HR 1.24, P = 0.0002), syndecan-1 (HR 1.27, P = 0.0002), suppression of tumorigenicity-2 (ST2) (IL1RL1; HR 1.22, P = 0.0002), interleukin (IL)-8 (CXCL8; HR 1.22, P = 0.0005), pentraxin (PTX)-3 (HR 1.17, P = 0.001), neutrophil gelatinase-associated lipocalin (LCN2; HR 1.18, P < 0.0001), osteoprotegerin (OPG) (TNFRSF11B; HR 1.26, P = 0.0002), and endostatin (COL18A1; HR 1.28, P = 0.0012). Several biomarkers were also significantly predictive of DHFA in adjusted analyses including FGF-23 (HR 1.36, P < 0.0001), TNF-α (HR 1.26, P < 0.0001), TNFR1 (HR 1.34, P < 0.0001), angiopoietin-2 (HR 1.26, P < 0.0001), syndecan-1 (HR 1.23, P = 0.0006), ST2 (HR 1.27, P < 0.0001), IL-8 (HR 1.18, P = 0.0009), PTX-3 (HR 1.18, P = 0.0002), OPG (HR 1.20, P = 0.0013), and NT-proBNP (HR 1.63, P < 0.0001). Machine-learning multimarker models were strongly associated with adverse outcomes (mean 1-year probability of death of 0%, 2%, and 60%; mean 1-year probability of DHFA of 0%, 4%, 97%; P < 0.0001). In these models, IL-6 (a biomarker of inflammation) and FGF-23 (a biomarker of calcification) emerged as the biomarkers of highest importance. CONCLUSIONS: Plasma biomarkers are strongly associated with the risk of adverse outcomes in patients with AS. Biomarkers of inflammation and calcification were most strongly related to prognosis.

Discovery of BMS-753426: A Potent Orally Bioavailable Antagonist of CC Chemokine Receptor 2.

To improve the metabolic stability profile of BMS-741672 (1a), we undertook a structure-activity relationship study in our trisubstituted cyclohexylamine series. This ultimately led to the identification of 2d (BMS-753426) as a potent and orally bioavailable antagonist of CCR2. Compared to previous clinical candidate 1a, the tert-butyl amine 2d showed significant improvements in pharmacokinetic properties, with lower clearance and higher oral bioavailability. Furthermore, compound 2d exhibited improved affinity for CCR5 and good activity in models of both monocyte migration and multiple sclerosis in the hCCR2 knock-in mouse. The synthesis of 2d was facilitated by the development of a simplified approach to key intermediate (4R)-9b that deployed a stereoselective reductive amination which may prove to be of general interest.

Integrin αVß1 regulates procollagen I production through a non-canonical transforming growth factor ß signaling pathway in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor ß (TGF-ß), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVß1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-ß1-activated primary human HSCs. Unexpectedly, either a selective αVß1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-ß1-activated procollagen I production in primary human HSCs, in which the role of ß1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-ß1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-ß-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVß1 integrin is involved in non-canonical TGF-ß signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-ß1 induced phosphorylation of ERK1/2 and decreased TGF-ß1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-ß1 induced procollagen I production. Taken together, current data indicate that αVß1 integrin can regulate TGF-ß signaling independent of its reported role in activating latent TGF-ß. Our data further support that αVß1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.

Bicyclic Ligand-Biased Agonists of S1P1: Exploring Side Chain Modifications to Modulate the PK, PD, and Safety Profiles.

Sphingosine-1-phosphate (S1P) binds to a family of sphingosine-1-phosphate G-protein-coupled receptors (S1P1-5). The interaction of S1P with these S1P receptors has a fundamental role in many physiological processes in the vascular and immune systems. Agonist-induced functional antagonism of S1P1 has been shown to result in lymphopenia. As a result, agonists of this type hold promise as therapeutics for autoimmune disorders. The previously disclosed differentiated S1P1 modulator BMS-986104 (1) exhibited improved preclinical cardiovascular and pulmonary safety profiles as compared to earlier full agonists of S1P1; however, it demonstrated a long pharmacokinetic half-life (T1/2 18 days) in the clinic and limited formation of the desired active phosphate metabolite. Optimization of this series through incorporation of olefins, ethers, thioethers, and glycols into the alkyl side chain afforded an opportunity to reduce the projected human T1/2 and improve the formation of the active phosphate metabolite while maintaining efficacy as well as the improved safety profile. These efforts led to the discovery of 12 and 24, each of which are highly potent, biased agonists of S1P1. These compounds not only exhibited shorter in vivo T1/2 in multiple species but are also projected to have significantly shorter T1/2 values in humans when compared to our first clinical candidate. In models of arthritis, treatment with 12 and 24 demonstrated robust efficacy.

Body Composition, Natriuretic Peptides, and Adverse Outcomes in Heart Failure With Preserved and Reduced Ejection Fraction.

OBJECTIVES: The purpose of this study was to determine the relationship between body composition, N-terminal B-type natriuretic peptide (NT-proBNP) levels, and heart failure (HF) phenotypes and outcomes. BACKGROUND: Abnormalities in body composition can influence metabolic dysfunction and HF severity; however, data assessing fat distribution and skeletal muscle (SM) size in HF with reduced (HFrEF) and preserved EF (HFpEF) are limited. Further, whether NPs relate more closely to axial muscle mass than measures of adiposity is not well studied. METHODS: We studied 572 adults without HF (n = 367), with HFrEF (n = 113), or with HFpEF (n = 92). Cardiac magnetic resonance was used to assess subcutaneous and visceral abdominal fat, paracardial fat, and axial SM size. We measured NT-proBNP in 334 participants. We used Cox regression to analyze the relationship between body composition and mortality. RESULTS: Compared with controls, pericardial and subcutaneous fat thickness were significantly increased in HFpEF, whereas patients with HFrEF had reduced axial SM size after adjusting for age, sex, race, and body height (p < 0.05 for comparisons). Lower axial SM size, but not fat, was significantly predictive of death in unadjusted (standardized hazard ratio: 0.63; p < 0.0001) and multivariable-adjusted analyses (standardized hazard ratio = 0.72; p = 0.0007). NT-proBNP levels more closely related to lower axial SM rather than fat distribution or body mass index (BMI) in network analysis, and when simultaneously assessed, only SM (p = 0.0002) but not BMI (p = 0.18) was associated with NT-proBNP. However, both NT-proBNP and axial SM mass were independently predictive of death (p < 0.05). CONCLUSIONS: HFpEF and HFrEF have distinct abnormalities in body composition. Reduced axial SM, but not fat, independently predicts mortality. Greater axial SM more closely associates with lower NT-proBNP rather than adiposity. Lower NT-proBNP levels in HFpEF compared with HFrEF relate more closely to muscle mass rather than obesity.

Pharmacodynamics-based approach for efficacious human dose projection of BMS-986260, a small molecule transforming growth factor beta receptor 1 inhibitor.

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine that has a wide array of biological effects. For decades, tumor biology implicated TGF-ß as an attractive therapeutic target due to its immunosuppressive effects. Toward this end, multiple pharmaceutical companies developed a number of drug modalities that specifically target the TGF-ß pathway. BMS-986260 is a small molecule, selective TGF-ßR1 kinase inhibitor that was under preclinical development for oncology. In vivo studies across mouse, rat, dog, and monkey and cryopreserved hepatocytes predicted human pharmacokinetics (PK) and distribution of BMS-986260. Efficacy studies of BMS-986260 were undertaken in the MC38 murine colon cancer model, and target engagement, as measured by phosphorylation of SMAD2/3, was assessed in whole blood to predict the clinical efficacious dose. The human clearance is predicted to be low, 4.25 ml/min/kg. BMS-986260 provided a durable and robust antitumor response at 3.75 mg/kg daily and 1.88 mg/kg twice-daily dosing regimens. Phosphorylation of SMAD2/3 was 3.5-fold less potent in human monocytes than other preclinical species. Taken together, the projected clinical efficacious dose was 600 mg QD or 210 mg BID for 3 days followed by a 4-day drug holiday. Mechanism-based cardiovascular findings in the rat ultimately led to the termination of BMS-986260. This study describes the preclinical PK characterization and pharmacodynamics-based efficacious dose projection of a novel small molecule TGF-ßR1 inhibitor.

Enabling direct and definitive free fraction determination for highly-bound compounds in protein binding assay.

Protein binding determination for highly-bound compounds using equilibrium dialysis remains a challenge in drug discovery. The reasons are mainly three-fold; 1. due to their slow diffusion rate, highly-bound compounds require a much longer incubation time to reach dialysis equilibrium than typically needed; 2. highly-bound compounds are often hydrophobic and prone to non-specific binding in dialysis; 3. free drug concentration in the post incubation dialysate is too low for reliable analytical quantification. Modified equilibrium dialysis approaches include using diluted plasma for dialysis, or pre-saturating the non-specific binding sites in the dialysis device with compounds of interest prior to dialysis. In this study, we developed a customized equilibrium dialysis assay with an extended incubation time of 24 h, followed by microflow (µF) LC-MS/MS for bioanalysis, for direct and definitive free fraction determination of highly protein-bound compounds. The extended incubation time ensured the dialysis to reach equilibrium and saturating the non-specific binding sites, while µFLC-MS/MS provided far better sensitivity than the conventional LC-MS/MS typically used for post incubation bioanalysis. For a group of commercially available, highly protein-bound compounds, the free fraction data generated by the developed assay correlated very well with the literature values generated with diluted plasma method or pre-saturation method. This novel assay approach has been successfully used to generate protein binding results for highly-bound compounds to support ongoing drug discovery research.

Aryl Ether-Derived Sphingosine-1-Phosphate Receptor (S1P1) Modulators: Optimization of the PK, PD, and Safety Profiles.

Efforts aimed at increasing the in vivo potency and reducing the elimination half-life of 1 and 2 led to the identification of aryl ether and thioether-derived bicyclic S1P1 differentiated modulators 3-6. The effects of analogs 3-6 on lymphocyte reduction in the rat (desired pharmacology) along with pulmonary- and cardiovascular-related effects (undesired pharmacology) are described. Optimization of the overall properties in the aryl ether series yielded 3d, and the predicted margin of safety against the cardiovascular effects of 3d would be large enough for human studies. Importantly, compared to 1 and 2, compound 3d had a better profile in both potency (ED50 < 0.05 mg/kg) and predicted human half-life (t 1/2 ∼ 5 days).

Quantifying drug tissue biodistribution by integrating high content screening with deep-learning analysis.

Quantitatively determining in vivo achievable drug concentrations in targeted organs of animal models and subsequent target engagement confirmation is a challenge to drug discovery and translation due to lack of bioassay technologies that can discriminate drug binding with different mechanisms. We have developed a multiplexed and high-throughput method to quantify drug distribution in tissues by integrating high content screening (HCS) with U-Net based deep learning (DL) image analysis models. This technology combination allowed direct visualization and quantification of biologics drug binding in targeted tissues with cellular resolution, thus enabling biologists to objectively determine drug binding kinetics.