Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors.

Autophagy is postulated to be required by cancer cells to survive periods of metabolic and/or hypoxic stress. ATG7 is the E1 enzyme that is required for activation of Ubl conjugation pathways involved in autophagosome formation. This article describes the design and optimization of pyrazolopyrimidine sulfamate compounds as potent and selective inhibitors of ATG7. Cellular levels of the autophagy markers, LC3B and NBR1, are regulated following treatment with these compounds.

Optimization of tetrahydronaphthalene inhibitors of Raf with selectivity over hERG.

Investigations of a biaryl ether scaffold identified tetrahydronaphthalene Raf inhibitors with good in vivo activity; however these compounds had affinity toward the hERG potassium channel. Herein we describe our work to eliminate this hERG activity via alteration of the substituents on the benzoic amide functionality. The resulting compounds have improved selectivity against the hERG channel, good pharmacokinetic properties and potently inhibit the Raf pathway in vivo.

Machine Learning Methods Using Artificial Intelligence Deployed on Electronic Health Record Data for Identification and Referral of At-Risk Patients From Primary Care Physicians to Eye Care Specialists: Retrospective, Case-Controlled Study.

BACKGROUND: Identification and referral of at-risk patients from primary care practitioners (PCPs) to eye care professionals remain a challenge. Approximately 1.9 million Americans suffer from vision loss as a result of undiagnosed or untreated ophthalmic conditions. In ophthalmology, artificial intelligence (AI) is used to predict glaucoma progression, recognize diabetic retinopathy (DR), and classify ocular tumors; however, AI has not yet been used to triage primary care patients for ophthalmology referral. OBJECTIVE: This study aimed to build and compare machine learning (ML) methods, applicable to electronic health records (EHRs) of PCPs, capable of triaging patients for referral to eye care specialists. METHODS: Accessing the Optum deidentified EHR data set, 743,039 patients with 5 leading vision conditions (age-related macular degeneration [AMD], visually significant cataract, DR, glaucoma, or ocular surface disease [OSD]) were exact-matched on age and gender to 743,039 controls without eye conditions. Between 142 and 182 non-ophthalmic parameters per patient were input into 5 ML methods: generalized linear model, L1-regularized logistic regression, random forest, Extreme Gradient Boosting (XGBoost), and J48 decision tree. Model performance was compared for each pathology to select the most predictive algorithm. The area under the curve (AUC) was assessed for all algorithms for each outcome. RESULTS: XGBoost demonstrated the best performance, showing, respectively, a prediction accuracy and an AUC of 78.6% (95% CI 78.3%-78.9%) and 0.878 for visually significant cataract, 77.4% (95% CI 76.7%-78.1%) and 0.858 for exudative AMD, 79.2% (95% CI 78.8%-79.6%) and 0.879 for nonexudative AMD, 72.2% (95% CI 69.9%-74.5%) and 0.803 for OSD requiring medication, 70.8% (95% CI 70.5%-71.1%) and 0.785 for glaucoma, 85.0% (95% CI 84.2%-85.8%) and 0.924 for type 1 nonproliferative diabetic retinopathy (NPDR), 82.2% (95% CI 80.4%-84.0%) and 0.911 for type 1 proliferative diabetic retinopathy (PDR), 81.3% (95% CI 81.0%-81.6%) and 0.891 for type 2 NPDR, and 82.1% (95% CI 81.3%-82.9%) and 0.900 for type 2 PDR. CONCLUSIONS: The 5 ML methods deployed were able to successfully identify patients with elevated odds ratios (ORs), thus capable of patient triage, for ocular pathology ranging from 2.4 (95% CI 2.4-2.5) for glaucoma to 5.7 (95% CI 5.0-6.4) for type 1 NPDR, with an average OR of 3.9. The application of these models could enable PCPs to better identify and triage patients at risk for treatable ophthalmic pathology. Early identification of patients with unrecognized sight-threatening conditions may lead to earlier treatment and a reduced economic burden. More importantly, such triage may improve patients' lives.

TAK-676: A Novel Stimulator of Interferon Genes (STING) Agonist Promoting Durable IFN-dependent Antitumor Immunity in Preclinical Studies.

Oncology therapies targeting the immune system have improved patient outcomes across a wide range of tumor types, but resistance due to an inadequate T-cell response in a suppressive tumor microenvironment (TME) remains a significant problem. New therapies that activate an innate immune response and relieve this suppression may be beneficial to overcome this hurdle. TAK-676 is a synthetic novel stimulator of interferon genes (STING) agonist designed for intravenous administration. Here we demonstrate that TAK-676 dose-dependently triggers activation of the STING signaling pathway and activation of type I interferons. Furthermore, we show that TAK-676 is a highly potent modulator of both the innate and adaptive immune system and that it promotes the activation of dendritic cells, natural killer cells, and T cells in preclinical models. In syngeneic murine tumor models in vivo, TAK-676 induces dose-dependent cytokine responses and increases the activation and proliferation of immune cells within the TME and tumor-associated lymphoid tissue. We also demonstrate that TAK-676 dosing results in significant STING-dependent antitumor activity, including complete regressions and durable memory T-cell immunity. We show that TAK-676 is well tolerated, exhibits dose-proportional pharmacokinetics in plasma, and exhibits higher exposure in tumor. The intravenous administration of TAK-676 provides potential treatment benefit in a broad range of tumor types. Further study of TAK-676 in first-in-human phase I trials is ongoing. Significance: TAK-676 is a novel systemic STING agonist demonstrating robust activation of innate and adaptive immune activity resulting in durable antitumor responses within multiple syngeneic tumor models. Clinical investigation of TAK-676 is ongoing.

Discovery and optimization of N-acyl and N-aroylpyrazolines as B-Raf kinase inhibitors.

A high throughput screen identified N-aroylpyrazoline 1 as a selective inhibitor of the V600E mutant of B-Raf kinase. Parallel synthesis of acyl, aroyl, and sulfonyl derivatives led to the identification of several potent inhibitors in both enzymatic and cellular (pERK) assays such as compound 42.

Discovery and optimization of pyrazoline compounds as B-Raf inhibitors.

The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.

Automated High-Throughput Flow Cytometry for High-Content Screening in Antibody Development.

The tedious sample preparation for flow cytometry limits the throughput and thus its usage as a primary screening method despite its sensitivity and accuracy. With the growing focus on utilizing antibodies as a therapeutic modality in drug discovery, it is critical to develop a high-throughput flow cytometry (HTFC) workflow to cope with the increasing need to support antibody discovery programs. We have developed a seamless HTFC sample preparation and readout workflow using the HighRes modular robotic system and the IntelliCyt iQue Screener PLUS. To fully utilize the advantages offered by flow cytometry, we typically multiplex multiple cell lines of interest in one well to simultaneously quantitate on-target activity and nonspecific activity along with measurement of antibody concentration. The ability to measure multiple parameters coupled with speed and increased accuracy provides gains in productivity and helps speed up antibody lead discovery.

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment.

The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

Design and optimization of potent and orally bioavailable tetrahydronaphthalene Raf inhibitors.

Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.

Genome-wide siRNA screen for modulators of cell death induced by proteasome inhibitor bortezomib.

Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.

ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis.

The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH + E zinc-binding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2- and 3.0-A resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other ( approximately 16 degrees ) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-[1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino]-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S(2)' substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2.