Computational analysis of kinase inhibitor selectivity using structural knowledge.

Motivation: Kinases play a significant role in diverse disease signaling pathways and understanding kinase inhibitor selectivity, the tendency of drugs to bind to off-targets, remains a top priority for kinase inhibitor design and clinical safety assessment. Traditional approaches for kinase selectivity analysis using biochemical activity and binding assays are useful but can be costly and are often limited by the kinases that are available. On the other hand, current computational kinase selectivity prediction methods are computational intensive and can rarely achieve sufficient accuracy for large-scale kinome wide inhibitor selectivity profiling. Results: Here, we present a KinomeFEATURE database for kinase binding site similarity search by comparing protein microenvironments characterized using diverse physiochemical descriptors. Initial selectivity prediction of 15 known kinase inhibitors achieved an >90% accuracy and demonstrated improved performance in comparison to commonly used kinase inhibitor selectivity prediction methods. Additional kinase ATP binding site similarity assessment (120 binding sites) identified 55 kinases with significant promiscuity and revealed unexpected inhibitor cross-activities between PKR and FGFR2 kinases. Kinome-wide selectivity profiling of 11 kinase drug candidates predicted novel as well as experimentally validated off-targets and suggested structural mechanisms of kinase cross-activities. Our study demonstrated potential utilities of our approach for large-scale kinase inhibitor selectivity profiling that could contribute to kinase drug development and safety assessment. Availability and implementation: The KinomeFEATURE database and the associated scripts for performing kinase pocket similarity search can be downloaded from the Stanford SimTK website (https://simtk.org/projects/kdb). Supplementary information: Supplementary data are available at Bioinformatics online.

Investigation of the absolute bioavailability and human mass balance of navoximod, a novel IDO1 inhibitor.

AIMS: Navoximod (GDC-0919, NLG-919) is a small molecule inhibitor of indoleamine-2,3-dioxygenase 1 (IDO1), developed to treat the acquired immune tolerance associated with cancer. The primary objectives of this study were to assess navoximod's absolute bioavailability (aBA), determine the mass balance and routes of elimination of [14 C]-navoximod, and characterize navoximod's metabolite profile. METHODS: A phase 1, open-label, two-part study was conducted in healthy volunteers. In Part 1 (aBA), subjects (n = 16) were randomized to receive oral (200 mg tablet) or intravenous (5 mg solution) navoximod in a crossover design with a 5-day washout. In Part 2 (mass balance), subjects (n = 8) were administered [14 C]-navoximod (200 mg/600 µCi) as an oral solution. RESULTS: The aBA of navoximod was estimated to be 55.5%, with a geometric mean (%CV) plasma clearance and volume of distribution of 62.0 L/h (21.0%) and 1120 L (28.4%), respectively. Mean recovery of total radioactivity was 87.8%, with 80.4% detected in urine and the remainder (7.4%) in faeces. Navoximod was extensively metabolized, with unchanged navoximod representing 5.45% of the dose recovered in the urine and faeces. Glucuronidation was identified as the primary route of metabolism, with the major glucuronide metabolite, M28, accounting for 57.5% of the total drug-derived exposure and 59.7% of the administered dose recovered in urine. CONCLUSIONS: Navoximod was well tolerated, quickly absorbed and showed moderate bioavailability, with minimal recovery of the dose as unchanged parent in the urine and faeces. Metabolism was identified as the primary route of clearance and navoximod glucuronide (M28) was the most abundant metabolite in circulation with all other metabolites accounting for <10% of drug-related exposure.

A phase Ib study of pictilisib (GDC-0941) in combination with paclitaxel, with and without bevacizumab or trastuzumab, and with letrozole in advanced breast cancer.

BACKGROUND: This phase Ib study (NCT00960960) evaluated pictilisib (GDC-0941; pan-phosphatidylinositol 3-kinase inhibitor) plus paclitaxel, with and without bevacizumab or trastuzumab, or in combination with letrozole, in patients with locally recurrent or metastatic breast cancer. METHODS: This was a three-part multischedule study. Patients in parts 1 and 2, which comprised 3 + 3 dose escalation and cohort expansion stages, received pictilisib (60-330 mg) plus paclitaxel (90 mg/m2) with and without bevacizumab (10 mg/kg) or trastuzumab (2-4 mg/kg). In part 3, patients received pictilisib (260 mg) plus letrozole (2.5 mg). Primary objectives were evaluation of safety and tolerability, identification of dose-limiting toxicities (DLTs) and the maximum tolerated dose (MTD) of pictilisib, and recommendation of a phase II dosing regimen. Secondary endpoints included pharmacokinetics and preliminary antitumor activity. RESULTS: Sixty-nine patients were enrolled; all experienced at least one adverse event (AE). Grade ≥ 3 AEs, serious AEs, and AEs leading to death were reported in 50 (72.5%), 21 (30.4%), and 2 (2.9%) patients, respectively. Six (8.7%) patients reported a DLT, and the MTD and recommended phase II pictilisib doses were established where possible. There was no pictilisib-paclitaxel drug-drug interaction. Two (3.4%) patients experienced complete responses, and 17 (29.3%) patients had partial responses. CONCLUSIONS: Combining pictilisib with paclitaxel, with and without bevacizumab or trastuzumab, or letrozole, had a manageable safety profile in patients with locally recurrent or metastatic breast cancer. The combination had antitumor activity, and the additive effect of pictilisib supported further investigation in a randomized study. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00960960 . Registered on August 13, 2009.

Genome-wide discovery of drug-dependent human liver regulatory elements.

Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements.

OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin.

Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.

Renal transporters in drug development.

The kidney plays a vital role in the body's defense against potentially toxic xenobiotics and metabolic waste products through elimination pathways. In particular, secretory transporters in the proximal tubule are major determinants of the disposition of xenobiotics, including many prescription drugs. In the past decade, considerable progress has been made in understanding the impact of renal transporters on the disposition of many clinically used drugs. In addition, renal transporters have been implicated as sites for numerous clinically important drug-drug interactions. This review begins with a description of renal drug handling and presents relevant equations for the calculation of renal clearance, including filtration and secretory clearance. In addition, data on the localization, expression, substrates, and inhibitors of renal drug transporters are tabulated. The recent US Food and Drug Administration drug-drug interaction draft guidance as it pertains to the study of renal drug transporters is presented. Renal drug elimination in special populations and transporter splicing variants are also described.

Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors.

To enhance the therapeutic index of T-cell engagers (TCEs), we engineered masked, precision-activated TCEs (XPAT proteins), targeting a tumor antigen (human epidermal growth factor receptor 2 (HER2) or epidermal growth factor receptor (EGFR)) and CD3. Unstructured XTEN polypeptide masks flank the N and C termini of the TCE and are designed to be released by proteases in the tumor microenvironment. In vitro, unmasked HER2-XPAT (uTCE) demonstrates potent cytotoxicity, with XTEN polypeptide masking providing up to 4-log-fold protection. In vivo, HER2-XPAT protein induces protease-dependent antitumor activity and is proteolytically stable in healthy tissues. In non-human primates, HER2-XPAT protein demonstrates a strong safety margin (>400-fold increase in tolerated maximum concentration versus uTCE). HER2-XPAT protein cleavage is low and similar in plasma samples from healthy and diseased humans and non-human primates, supporting translatability of stability to patients. EGFR-XPAT protein confirmed the utility of XPAT technology for tumor targets more widely expressed in healthy tissues.

First-in-Human Phase I Study to Evaluate the Brain-Penetrant PI3K/mTOR Inhibitor GDC-0084 in Patients with Progressive or Recurrent High-Grade Glioma.

PURPOSE: GDC-0084 is an oral, brain-penetrant small-molecule inhibitor of PI3K and mTOR. A first-in-human, phase I study was conducted in patients with recurrent high-grade glioma. PATIENTS AND METHODS: GDC-0084 was administered orally, once daily, to evaluate safety, pharmacokinetics (PK), and activity. Fluorodeoxyglucose-PET (FDG-PET) was performed to measure metabolic responses. RESULTS: Forty-seven heavily pretreated patients enrolled in eight cohorts (2-65 mg). Dose-limiting toxicities included 1 case of grade 2 bradycardia and grade 3 myocardial ischemia (15 mg), grade 3 stomatitis (45 mg), and 2 cases of grade 3 mucosal inflammation (65 mg); the MTD was 45 mg/day. GDC-0084 demonstrated linear and dose-proportional PK, with a half-life (∼19 hours) supportive of once-daily dosing. At 45 mg/day, steady-state concentrations exceeded preclinical target concentrations producing antitumor activity in xenograft models. FDG-PET in 7 of 27 patients (26%) showed metabolic partial response. At doses ≥45 mg/day, a trend toward decreased median standardized uptake value in normal brain was observed, suggesting central nervous system penetration of drug. In two resection specimens, GDC-0084 was detected at similar levels in tumor and brain tissue, with a brain tissue/tumor-to-plasma ratio of >1 and >0.5 for total and free drug, respectively. Best overall response was stable disease in 19 patients (40%) and progressive disease in 26 patients (55%); 2 patients (4%) were nonevaluable. CONCLUSIONS: GDC-0084 demonstrated classic PI3K/mTOR-inhibitor related toxicities. FDG-PET and concentration data from brain tumor tissue suggest that GDC-0084 crossed the blood-brain barrier.

Alternative dosing regimens for atezolizumab: an example of model-informed drug development in the postmarketing setting.

PURPOSE: To determine the exposure-response (ER) relationships between atezolizumab exposure and efficacy or safety in patients with advanced non-small cell lung cancer (NSCLC) or urothelial carcinoma (UC) and to identify alternative dosing regimens. METHODS: ER analyses were conducted using pooled NSCLC and UC data from phase 1 and 3 studies (PCD4989g, OAK, IMvigor211; ClinicalTrials.gov IDs, NCT01375842, NCT02008227, and NCT02302807, respectively). Objective response rate, overall survival, and adverse events were evaluated vs pharmacokinetic (PK) metrics. Population PK-simulated exposures for regimens of 840 mg every 2 weeks (q2w) and 1680 mg every 4 weeks (q4w) were compared with the approved regimen of 1200 mg every 3 weeks (q3w) and the maximum assessed dose (MAD; 20 mg/kg q3w). Phase 3 IMpassion130 (NCT02425891) data were used to validate the PK simulations for 840 mg q2w. Observed safety data were evaluated by exposure and body weight subgroups. RESULTS: No significant ER relationships were observed for safety or efficacy. Predicted exposures for 840 mg q2w and 1680 mg q4w were comparable to 1200 mg q3w and the MAD and consistent with observed PK data from IMpassion130. Observed safety was similar between patients with a Cmax above and below the predicted Cmax for 1680 mg q4w and between patients in the lowest and upper 3 body weight quartiles. CONCLUSION: Atezolizumab regimens of 840 mg q2w and 1680 mg q4w are expected to have comparable efficacy and safety as the approved regimen of 1200 mg q3w, supporting their interchangeable use and offering patients greater flexibility.

Phase Ia study of the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor navoximod (GDC-0919) in patients with recurrent advanced solid tumors.

BACKGROUND: Indoleamine-2,3-dioxygenase 1 (IDO1) catalyzes the oxidation of tryptophan into kynurenine and is partially responsible for acquired immune tolerance associated with cancer. The IDO1 small molecule inhibitor navoximod (GDC-0919, NLG-919) is active as a combination therapy in multiple tumor models. METHODS: This open-label Phase Ia study assessed safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary anti-tumor activity of navoximod in patients with recurrent/advanced solid tumors, administered as 50-800 mg BID on a 21/28 day and at 600 mg on a 28/28 day schedule. Plasma kynurenine and tryptophan were longitudinally evaluated and tumor assessments were performed. RESULTS: Patients (n = 22) received a median of 3 cycles of navoximod. No maximum tolerated dose was reached. One dose-limiting toxicity of Grade 4 lower gastrointestinal hemorrhage was reported. Adverse events (AEs) regardless of causality in ≥20% of patients included fatigue (59%), cough, decreased appetite, and pruritus (41% each), nausea (36%), and vomiting (27%). Grade ≥ 3 AEs occurred in 14/22 patients (64%), and were related to navoximod in two patients (9%). Navoximod was rapidly absorbed (Tmax ~ 1 h) and exhibited dose-proportional increases in exposure, with a half-life (t1/2 ~ 11 h) supportive of BID dosing. Navoximod transiently decreased plasma kynurenine from baseline levels with kinetics consistent with its half-life. Of efficacy-evaluable patients, 8 (36%) had stable disease and 10 (46%) had progressive disease. CONCLUSIONS: Navoximod was well-tolerated at doses up to 800 mg BID decreasing plasma kynurenine levels consistent with its half-life. Stable disease responses were observed. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02048709 .

Combining "Bottom-up" and "Top-down" Approaches to Assess the Impact of Food and Gastric pH on Pictilisib (GDC-0941) Pharmacokinetics.

Pictilisib, a weakly basic compound, is an orally administered, potent, and selective pan-inhibitor of phosphatidylinositol 3-kinases for oncology indications. To investigate the significance of high-fat food and gastric pH on pictilisib pharmacokinetics (PK) and enable label recommendations, a dedicated clinical study was conducted in healthy volunteers, whereby both top-down (population PK, PopPK) and bottom-up (physiologically based PK, PBPK) approaches were applied to enhance confidence of recommendation and facilitate the clinical development through scenario simulations. The PopPK model identified food (for absorption rate constant (Ka )) and proton pump inhibitors (PPI, for relative bioavailability (Frel ) and Ka ) as significant covariates. Food and PPI also impacted the variability of Frel . The PBPK model accounted for the supersaturation tendency of pictilisib, and gastric emptying physiology successfully predicted the food and PPI effect on pictilisib absorption. Our research highlights the importance of applying both quantitative approaches to address critical drug development questions.

The Effect of Nizatidine, a MATE2K Selective Inhibitor, on the Pharmacokinetics and Pharmacodynamics of Metformin in Healthy Volunteers.

BACKGROUND AND OBJECTIVES: In the proximal tubule, basic drugs are transported from the renal cells to the tubule lumen through the concerted action of the H(+)/organic cation antiporters, multidrug and toxin extrusion (MATE) 1 and MATE2K. Dual inhibitors of the MATE transporters have been shown to have a clinically relevant effect on the pharmacokinetics of concomitantly administered basic drugs. However, the clinical impact of selective renal organic cation transport inhibition on the pharmacokinetics and pharmacodynamics of basic drugs, such as metformin, is unknown. This study sought to identify a selective MATE2K inhibitor in vitro and to determine its clinical impact on the pharmacokinetics and pharmacodynamics of metformin in healthy subjects. METHODS: Strategic cell-based screening of 71 US Food and Drug Administration (FDA)-approved medications was conducted to identify selective inhibitors of renal organic cation transporters that are capable of inhibiting at clinically relevant concentrations. From this screen, nizatidine was identified and predicted to be a clinically potent and selective inhibitor of MATE2K-mediated transport. The effect of nizatidine on the pharmacokinetics and pharmacodynamics of metformin was evaluated in 12 healthy volunteers in an open-label, randomized, two-phase crossover drug-drug interaction (DDI) study. RESULTS: In healthy volunteers, the MATE2K-selective inhibitor nizatidine significantly increased the apparent volume of distribution, half-life, and hypoglycemic activity of metformin. However, despite achieving unbound maximum concentrations greater than the in vitro inhibition potency (concentration of drug producing 50% inhibition [IC50]) of MATE2K-mediated transport, nizatidine did not affect the renal clearance (CLR) or net secretory clearance of metformin. CONCLUSION: This study demonstrates that a selective inhibition of MATE2K by nizatidine affected the apparent volume of distribution, tissue concentrations, and peripheral effects of metformin. However, nizatidine did not alter systemic concentrations or the CLR of metformin, suggesting that specific MATE2K inhibition may not be sufficient to cause renal DDIs with metformin.

Discovery of potent, selective multidrug and toxin extrusion transporter 1 (MATE1, SLC47A1) inhibitors through prescription drug profiling and computational modeling.

The human multidrug and toxin extrusion (MATE) transporter 1 contributes to the tissue distribution and excretion of many drugs. Inhibition of MATE1 may result in potential drug-drug interactions (DDIs) and alterations in drug exposure and accumulation in various tissues. The primary goals of this project were to identify MATE1 inhibitors with clinical importance or in vitro utility and to elucidate the physicochemical properties that differ between MATE1 and OCT2 inhibitors. Using a fluorescence assay of ASP(+) uptake in cells stably expressing MATE1, over 900 prescription drugs were screened and 84 potential MATE1 inhibitors were found. We identified several MATE1 selective inhibitors including four FDA-approved medications that may be clinically relevant MATE1 inhibitors and could cause a clinical DDI. In parallel, a QSAR model identified distinct molecular properties of MATE1 versus OCT2 inhibitors and was used to screen the DrugBank in silico library for new hits in a larger chemical space.

Expanding the role of the dynein regulatory complex to non-axonemal functions: association of GAS11 with the Golgi apparatus.

The mammalian GAS11 gene is a candidate tumor suppressor of unknown function that was previously identified as one of several genes upregulated upon growth arrest. Interestingly, although GAS11 homologs in Trypanosoma brucei (trypanin) and Chlamydomonas reinhardtii (PF2) are integral components of the flagellar axoneme and are necessary for regulating flagellar beat, the GAS11 gene was discovered based on its expression in cells that do not assemble a motile cilium. This suggests that GAS11 function might not be restricted to the cilium. To investigate this possibility, we generated GAS11-specific antibodies and demonstrate here that GAS11 is expressed in a variety of mammalian cells that lack a motile cilium. In COS7 cells, GAS11 is associated with the detergent-insoluble cytoskeleton and exhibits a juxtanuclear localization that overlaps with the pericentrosomal Golgi apparatus. This localization is dependent upon intact microtubules and is cell-cycle regulated, such that GAS11 is dispersed throughout the cytoplasm as cells progress through mitosis. GAS11 remains associated with Golgi fragments following depolymerization of cytoplasmic microtubules but is dispersed upon disruption of the Golgi with brefeldin A. These data suggest that GAS11 is associated with the Golgi apparatus. In support of this, recombinant GAS11 binds Golgi membranes in vitro. In growth-arrested mIMCD3 cells, GAS11 co-localizes with gamma-tubulin at the base of the primary cilium. The pericentrosomal Golgi apparatus and base of the cilium both represent convergence points for microtubule minus ends and correspond to sites where dynein regulation is required. The algal GAS11 homolog functions as part of a dynein regulatory complex (DRC) in the axoneme (Rupp and Porter. J Cell Biol 2003;162:47-57) and our findings suggest that components of this axonemal dynein regulatory system have been adapted in mammalian cells to participate in non-axonemal functions.