Combinatorial High-throughput Screening Research Articles

Combination p53 activation and BCL-xL/BCL-2 inhibition as a therapeutic strategy in high-risk and relapsed acute lymphoblastic leukemia.

Due to the rarity of TP53 mutations in acute lymphoblastic leukemia (ALL), p53 re-activation by antagonism of the p53-MDM2 interaction represents a potential therapeutic strategy for the majority of ALL. Here, we demonstrate the potent antileukemic activity of the MDM2 antagonist idasanutlin in high-risk and relapsed ex vivo coculture models of TP53 wildtype ALL (n = 40). Insufficient clinical responses to monotherapy MDM2 inhibitors in other cancers prompted us to explore optimal drugs for combination therapy. Utilizing high-throughput combination screening of 1971 FDA-approved and clinically advanced compounds, we identified BCL-xL/BCL-2 inhibitor navitoclax as the most promising idasanutlin combination partner. The idasanutlin-navitoclax combination was synergistically lethal to prognostically-poor, primary-derived and primary patient blasts in ex vivo coculture, and reduced leukemia burden in two very high-risk ALL xenograft models at drug concentrations safely attained in patients; in fact, the navitoclax plasma concentrations were equivalent to those attained in contemporary "low-dose" navitoclax clinical trials. We demonstrate a preferential engagement of cell death over G1 cell cycle arrest, mechanistically implicating MCL-1-binding pro-apoptotic sensitizer NOXA. The proposed combination of two clinical-stage compounds independently under clinical evaluation for ALL is of high clinical relevance and warrants consideration for the treatment of patients with high-risk and relapsed ALL.

Combinatorial High-Throughput Screening of Complex Polymeric Enzyme Immobilization Supports.

Recent advances have demonstrated the promise of complex multicomponent polymeric supports to enable supra-biological enzyme performance. However, the discovery of such supports has been limited by time-consuming, low-throughput synthesis and screening. Here, we describe a novel combinatorial and high-throughput platform that enables rapid screening of complex and heterogeneous copolymer brushes as enzyme immobilization supports, named combinatorial high-throughput enzyme support screening (CHESS). Using a 384-well plate format, we synthesized arrays of three-component polymer brushes in the microwells using photoactivated surface-initiated polymerization and immobilized enzymes in situ. The utility of CHESS to identify optimal immobilization supports under thermally and chemically denaturing conditions was demonstrated usingBacillus subtilisLipase A (LipA). The identification of supports with optimal compositions was validated by immobilizing LipA on polymer-brush-modified biocatalyst particles. We further demonstrated that CHESS could be used to predict the optimal composition of polymer brushes a priori for the previously unexplored enzyme, alkaline phosphatase (AlkP). Our findings demonstrate that CHESS represents a predictable and reliable platform for dramatically accelerating the search of chemical compositions for immobilization supports and further facilitates the discovery of biocompatible and stabilizing materials.

Arrest of mouse preterm labor until term delivery by combination therapy with atosiban and mundulone, a natural product with tocolytic efficacy

There is a lack of FDA-approved tocolytics for the management of preterm labor (PL). In prior drug discovery efforts, we identified mundulone and mundulone acetate (MA) as inhibitors of in vitro intracellular Ca2+-regulated myometrial contractility. In this study, we probed the tocolytic potential of these compounds using human myometrial samples and a mouse model of preterm birth. In a phenotypic assay, mundulone displayed greater efficacy, while MA showed greater potency and uterine-selectivity in the inhibition of intracellular-Ca2+ mobilization. Cell viability assays revealed that MA was significantly less cytotoxic. Organ bath and vessel myography studies showed that only mundulone exerted inhibition of myometrial contractions and that neither compounds affected vasoreactivity of ductus arteriosus. A high-throughput combination screen identified that mundulone exhibits synergism with two clinical-tocolytics (atosiban and nifedipine), and MA displayed synergistic efficacy with nifedipine. Of these combinations, mundulone+atosiban demonstrated a significant improvement in the in vitro therapeutic index compared to mundulone alone. The ex vivo and in vivo synergism of mundulone+atosiban was substantiated, yielding greater tocolytic efficacy and potency on myometrial tissue and reduced preterm birth rates in a mouse model of PL compared to each single agent. Treatment with mundulone after mifepristone administration dose-dependently delayed the timing of delivery. Importantly, mundulone+atosiban permitted long-term management of PL, allowing 71% dams to deliver viable pups at term (>day 19, 4–5 days post-mifepristone exposure) without visible maternal and fetal consequences. Collectively, these studies provide a strong foundation for the development of mundulone as a single or combination tocolytic for management of PL.

High-Throughput Synthesis and Machine Learning Assisted Design of Photodegradable Hydrogels.

Due to the large chemical space, the design of functional and responsive soft materials poses many challenges but also offers a wide range of opportunities in terms of the scope of possible properties. Herein, an experimental workflow for miniaturized combinatorial high-throughput screening of functional hydrogel libraries is reported. The data created from the analysis of the photodegradation process of more than 900 different types of hydrogel pads are used to train a machine learning model for automated decision making. Through iterative model optimization based on Bayesian optimization, a substantial improvement in response properties is achieved and thus expanded the scope of material properties obtainable within the chemical space of hydrogels in the study. It is therefore demonstrated that the potential of combining miniaturized high-throughput experiments with smart optimization algorithms for cost and time efficient optimization of materials properties.

MEK inhibition causes BIM stabilization and increased sensitivity to BCL-2 family member inhibitors in RAS-MAPK-mutated neuroblastoma.

Mutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma tumors and are associated with response to MEK inhibition in vitro. However, these inhibitors alone do not lead to tumor regression in vivo, indicating the need for combination therapy. Via high-throughput combination screening, we identified that the MEK inhibitor trametinib can be combined with BCL-2 family member inhibitors, to efficiently inhibit growth of neuroblastoma cell lines with RAS-MAPK mutations. Suppressing the RAS-MAPK pathway with trametinib led to an increase in pro-apoptotic BIM, resulting in more BIM binding to anti-apoptotic BCL-2 family members. By favoring the formation of these complexes, trametinib treatment enhances sensitivity to compounds targeting anti-apoptotic BCL-2 family members. In vitro validation studies confirmed that this sensitizing effect is dependent on an active RAS-MAPK pathway. In vivo combination of trametinib with BCL-2 inhibitors led to tumor inhibition in NRAS-mutant and NF1-deleted xenografts. Together, these results show that combining MEK inhibition with BCL-2 family member inhibition could potentially improve therapeutic outcomes for RAS-MAPK-mutated neuroblastoma patients.

Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells

SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir’s apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.

Combined inhibition of EZH2 and ATM is synthetic lethal in BRCA1-deficient breast cancer

BackgroundThe majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype.MethodsOur group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors.ResultsWe identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors.ConclusionTaken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.

Abstract 3892: High-throughput combination screen for identifying novel therapies against high-risk neuroblastoma

Abstract Neuroblastoma is a childhood solid tumor commonly located in the adrenal gland. High-risk neuroblastoma patients undergo multidrug and multi-interventional treatment but despite these efforts many patients develop resistance to chemotherapy and eventually relapse. Neuroblastoma patients face an urgent need of novel therapeutic strategies that could effectively combat the disease. In a recent study we identified kinesin spindle protein (KSP) as a highly effective target in high-risk neuroblastoma. Pharmacological inhibition of KSP resulted in complete regression of a subset of neuroblastoma subcutaneous PDX tumors and tumor growth delay in orthotopic PDXs (Hansson, Radke et al, Sci. Transl. Med 2020). The purpose of the present study is to identify novel synergistic drug combinations effective against neuroblastoma and investigate their translational potential. We designed, optimized, and performed a high-throughput combination screen using 3D tumor organoids that represent three MYCN-amplified neuroblastoma tumors. KSP inhibition was tested in combination with the FIMM drug set of 527 approved and investigational drugs. Screening was performed stepwise: • Step 1: High-throughput drug screening 527 drug combinations and machine learning prediction (DECREASE tool) of drug combination synergy based on minimal required input. • Step 2: Screening of 6x6 dose-response matrices of top 26 predicted hits in high-risk neuroblastoma models. • Step 3: Screening of 6x6 dose-response matrices of top 26 hits in CD34+ cord blood controls to identify compounds that have low toxicity towards proliferating fraction of hematopoietic cells. Overall, we identified seven drug candidates (P1-P7). They classify as: kinase inhibitors, differentiating modifiers, and one hormone therapy. Screened combinations were chosen based on selective efficacy (difference between anti-neuroblastoma efficacy and efficacy towards CD34+ cord blood controls). Identified drug combinations are common among three high-risk neuroblastoma models and have most synergistic area score (MSA) over five. We further plan to validate seven hits in neuroblastoma models in vitro, investigate MOA of the most promising combination and test it in PDX models in vivo. Citation Format: Katarzyna Radke, Karin Hansson, Jani Saarela, Aleksandr Ianevski, Philipp Ianevski, Aurélie Baudet, Mattias Magnusson, Daniel Bexell. High-throughput combination screen for identifying novel therapies against high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3892.

Abstract 5408: Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in the management of high-grade serous ovarian cancer (HGSOC). However, most HGSOC ultimately develops resistance to PARPi, leading to unmet medical needs for better therapeutic strategies. ATR-mediated G2/M cell cycle checkpoints are important for cell survival and DNA repair, especially in HGSOC due to its dysfunctional p53-mediated G1/S checkpoint, making them an attractive therapeutic target. However, only modest monotherapy activity of ATR inhibitors (ATRis) has been reported in patients with HGSOC, highlighting the need for combination treatment. We conducted a high-throughput drug combination screen of ATRi (ceralasertib) with 1,912 drugs in two PARPi-resistant BRCA2-mutant PEO1 (PEO1/OlaR and PEO1/OlaJR) HGSOC cell lines. In this screen, 234 drugs showed synergistic cytotoxicity with ATRi, including 31 (13.2%) cell cycle checkpoint inhibitors, 27 (11.5%) chemotherapeutic drugs, and 13 (5.6%) PI3K/AKT pathway inhibitors. We prioritized the PI3K/AKT pathway inhibitors for ATRi combination because the PI3K/AKT pathway is upregulated in >70% of HGSOC and associated with an aggressive phenotype. Moreover, PI3K/AKT signaling is found to be activated in HGSOC cells with PARPi resistance. We separately validated that an ATRi (ceralasertib < 1 μM) in combination with an AKT inhibitor (AKTi; capivasertib < 10 μM) yielded additive/synergistic cytotoxic effects (combination index < 1) in various HGSOC cell lines, including acquired and de novo PARPi-resistant (PEO1/OlaR, PEO1/OlaJR, and PEO4) and platinum-resistant HGSOC cell lines (OVCAR3), by XTT and colony formation assays. With clinically attainable concentrations of ATRi (1 μM) and AKTi (10 μM), the combination treatment increased cell apoptosis compared to ATRi (increased 2.1 to 95.2-fold; P < 0.001) or AKTi alone (increased 3.3 to 92.2-fold; P < 0.001) in both PARPi-sensitive and -resistant HGSOC cells. ATRi+AKTi treatment also showed greater DNA damage as evidenced by increased percentage of cells with ≥ 5 γH2AX foci relative to ATRi (increased 1.1 to 7.6-fold; not significant in PEO1 and P < 0.001 in PARPi-resistant cells) or AKTi (increased 2.3 to 6.2-fold; P < 0.001) in both PARPi-sensitive and -resistant cells. Furthermore, ATRi+AKTi treatment augmented replication stress as measured by increased phospho-RPA+/γH2AX+ populations compared with ATRi (increased 1.7 to 4.7-fold; P < 0.001) or AKTi (increased 3.7 to 12.0-fold; P < 0.001). Overall, our results suggest that dual inhibition of ATR and AKT pathways results in greater cell death by increasing DNA damage and replication stress in HGSOC cells with PARPi resistance. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Nitasha Gupta, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5408.

Abstract 1403: ROS metabolism and autophagy as potential targets to improve head and neck cancer treatment

Abstract Introduction: Cancer cells exhibit a high degree of plasticity to adapt the rapidly changing tumor microenvironment (TME), in part by altering cellular metabolic pathways at a faster and more efficient pace than normal cells. Cancer cell plasticity, with genetic and epigenetic alterations, promote the diversity of cancer cells contributing to heterogeneity within the tumor. Head and neck cancer (HNC) is a notable example of this diversity; HNC tumors harbor cells with a wide genetic and metabolic heterogeneity, including cancer stem cells (CSC). Despite current treatment approaches, more than 40% of HNC patients have disease recurrence. Thus, we developed a multimodal approach to effectively prevent the development of radio/drug-resistance. Experimental Procedures: Nuclear-localized mkate2 expressing HNC cell numbers detected for every 6 hours by using IncuCyte S3 instrument. Apoptotic cells detected via NucView 488 Caspase-3 substrate and ROS levels determined using CellROX green simultaneously with cell numbers in the IncuCyte S3 instrument. Synergy scores calculated according to viability in SynergyFinder 2.0 web-application. Results: Our preliminary data profiling of metabolic regulators as radiosensitizer indicate that auranofin (AUR, thioredoxin reductase inhibitor) and buthionine sulfoximine (BSO, γ-glutamylcysteine synthetase inhibitor), given in combination with radiation (RT+AUR+BSO): 1) significantly inhibit proliferation in HNC cell lines, 2) increase ROS accumulation, and 3) reduce the surviving fraction of cells in colony forming assays compared to individual/dual agent applications. Furthermore, we added an autophagy inhibitor, SAR405, in our treatment regimen because of the crosstalk between ROS levels and autophagy. Human (FaDu and UM-SCC-47) and murine (MOC1 and MOC2) HNC cell were treated with +/-AUR+/-BSO+/-SAR405 and high-throughput combinatorial screening of cell numbers obtained over time. The combination of AUR+BSO+SAR405 registered a higher synergy score than the double combinations. Furthermore, as compared to single or double drug applications, the AUR+BSO+SAR405 group had a greater apoptotic cell rate and ROS levels. Conclusion: We present here a high-throughput method to screen multi-drug synergy. Multi-modal approach is important to prevent recurrence by providing a broader effect on heterogenous cancer cells in tumor. Moreover, multi-drug combinations allow usage of lower doses of drugs effectively, hence lowers the treatment-related adverse effects. This approach indicated the possibility of HNC treatment by increasing the ROS levels beyond the containable threshold which is consistent with the recent findings about the alterations in redox metabolism, particularly upregulation in thioredoxin and glutathione pathways, in many cancer types. In vivo experiments are ongoing to evaluate the potential of this combination for clinical studies. Citation Format: Zafer Gurel, Qianyun Luo, Michael S. Luy, Randall J. Kimple. ROS metabolism and autophagy as potential targets to improve head and neck cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1403.

Computational Pipeline for Rational Drug Combination Screening in Patient-Derived Cells.

In many complex diseases, such as cancers, resistance to monotherapies easily occurs, and longer-term treatment responses often require combinatorial therapies as next-line regimens. However, due to a massive number of possible drug combinations to test, there is a need for systematic and rational approaches to finding safe and effective drug combinations for each individual patient. This protocol describes an ecosystem of computational methods to guide high-throughput combinatorial screening that help experimental researchers to identify optimal drug combinations in terms of synergy, efficacy, and/or selectivity for further preclinical and clinical investigation. The methods are demonstrated in the context of combinatorial screening in primary cells of leukemia patients, where the translational aim is to identify drug combinations that show not only high synergy but also maximal cancer-selectivity. The mechanism-agnostic and cost-effective computational methods are widely applicable to various cancer types, which are amenable to drug testing, as the computational methods take as input only the phenotypic measurements of a subset of drug combinations, without requiring target information or genomic profiles of the patient samples.

Synergistic Combination Activity of the Novel GSPT1 Degrader CC-90009 in Acute Myeloid Leukemia Models

Introduction: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD ®) agent that is a first-in-class degrader of translation termination factor G1 to S phase transition 1 (GSPT1). CC-90009 has demonstrated antileukemic activity as a single agent and is currently under investigation in patients with acute myeloid leukemia (AML; NCT02848001). Treatment with CC-90009 led to rapid reductions in peripheral and bone marrow blasts, and demonstrated preliminary promising efficacy, including several complete remissions, in patients with relapsed or refractory AML. Here, we describe the identification and preclinical activity of select anti-AML agents as potential combination partners of CC-90009 to further improve its efficacy and therapeutic index. Based on these results, the combination activity of CC-90009 with venetoclax (VEN)/azacitidine (AZA) is being evaluated in a phase 1/2 trial in patients with AML (NCT04336982).Methods: A high-throughput cell viability screen was performed to identify synergistic partners of CC-90009. AML cell lines (HL-60, HNT-34, KG-1, ML-2, NOMO-1, MOLM-13, MV4-11, F-36P, OCI-AML2, OCI-AML3) were treated with CC-90009 in combination with > 70 compounds, including standard anti-AML agents, tyrosine kinase inhibitors (TKIs), unfolded protein response inducers, transcription inhibitors, and epigenetic agents. MOLM-13 and MV4-11 are fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) cell lines. Hits were validated in a colony formation (CF) assay using primary AML cells and bone marrow mononuclear cells (BMMC) from healthy donors. Synergy of the combination partners with CC-90009 was further assessed in AML patient-derived xenograft (PDX) models. Synergy between the isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib and CC-90009 was evaluated in a TF-1 erythroleukemia cell line overexpressing IDH2 R140Q mutant, and an IDH2 R140Q PDX model, AM7577.Results: Our high-throughput combination screen revealed multiple TKIs, epigenetic agents, and pro-apoptotic agents as potential combination partners of CC-90009 in AML cell lines. FLT3 inhibitors, including sunitinib, pexidartinib, midostaurin, lestaurtinib, crenolanib, and gilteritinib, synergized with CC-90009 to reduce viability in FLT3-ITD AML cell lines MV4-11 and MOLM-13. Similarly, the B-cell lymphoma 2 (BCL2) inhibitor VEN potentiated CC-90009-induced apoptosis and accelerated cell-autonomous killing. Reduction in levels of MCL-1, an anti-apoptotic factor, by CC-90009 most likely contributed to the synergy with VEN.We prioritized the evaluation of FLT3, BCL2, and IDH2 inhibitors as partners of CC-90009. In CF assays, midostaurin enhanced the inhibitory effect of CC-90009 in primary AML cells, without augmenting the effect of CC-90009 in CD34+ BMMC from healthy donors. Similarly, VEN enhanced the reduction in CF by CC-90009 in AML patient-derived BMMC without exacerbating the decrease in CF by CC-90009 in BMMC from healthy donors.We characterized FLT3 inhibitor/CC-90009 and BCL2 inhibitor/AZA/CC-90009 combinations in a FLT3-ITD PDX murine model, PDX1. The FLT3 inhibitor quizartinib significantly prolonged survival when combined with CC-90009 compared with either agent alone (P < 0.001). Similarly, VEN/AZA/CC-90009 combination markedly extended survival compared with single agents or VEN/AZA doublets (P < 0.001).The synergy between CC-90009 and epigenetic modulators was validated and further characterized in customized cell differentiation assays. Addition of CC-90009 to enasidenib, a mutant IDH2 inhibitor, enhanced differentiation and killing of CD34+ stem and progenitor cells, and increased differentiated CD235a+ erythroblasts, compared with enasidenib alone in a TF-1 cell line overexpressing IDH2 R140Q. Enasidenib/CC-90009 combination treatment reduced CD45+ malignant populations and increased differentiated CD14+ cells, leading to significantly prolonged animal survival in an IDH2 R140Q PDX model, AM7577, compared with either agent alone (P < 0.0001).Conclusion: Using a high-throughput combination screen, we identified rational combination partners that synergize with CC-90009 in in vitro and in vivo AML models. Collectively, these results support the clinical evaluation of CC-90009 in combination with FLT3, BCL2, and IDH2 inhibitors to further improve treatment outcomes for patients with AML. DisclosuresPierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Yao: Bristol Myers Squibb: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months, Research Funding. Pace: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Wang: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flandin-Blety: Bristol Myers Squibb: Current Employment. Benitez: Bristol Myers Squibb: Current Employment. Guarinos: Bristol Myers Squibb: Current Employment. Hoffmann: Bristol Myers Squibb: Current Employment. Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Electrochemical Polymer Pen Lithography.

The development of a massively parallel lithographic technique called electrochemical polymer pen lithography is reported. Pyramidal pen arrays, consisting of more than 10000 hydrogel pens loaded with metal salts, are integrated into a three-electrode cell and used to locally reduce ions at each pen tip. This system enables high-throughput patterning of a variety of metallic inks (e.g., Ni2+ , Pt2+ , Ag+ ) on the nanometer to micrometer length scale. By incorporating a z-direction piezo actuator, the extension length and dwell time can be used to precisely define feature dimensions (210 to 10µm in width, and up to 900nm in height, thus far). Furthermore, by controlling the potential and precursor concentrations, more than one element can be simultaneously deposited, creating a new tool for the synthesis of alloy features, such as NiCo, which are relevant for catalysis. Importantly, this methodology enables fine control over feature size and composition in a single pattern, which may make it ultimately useful for rapid, high-throughput combinatorial screening of metallic features.

Titanium Complexes with Functional Alkoxido Ligands for Selective Ethylene Dimerization – A High Throughput Experimentation Approach

AbstractNew titanium complexes of general formula [Ti(OR)2(OiPr)2], containing functionalized alkoxido ligands, were developed for the selective catalytic dimerization of ethylene to 1‐butene using a combined High Throughput Screening (HTS) / Design of Experiment (DoE) approach. First, a library of 19 ligands was elaborated and a primary screening spotted the phosphorus‐functionalized alkoxido ligands as most promising. A second, more focused library containing 8 alkoxidophosphane ligands was then developed. A longer linear spacer between the alkoxido and the phosphorus functions, as in [Ti(19)2(OiPr)2], was found beneficial for this catalytic reaction. After identification of the best co‐catalyst (AlEt3) and co‐ligand (OnBu), final optimization of the reaction conditions was performed using a design of experiments (DoE) approach. The complex [Ti(19)2(OnBu)2] was shown to selectively dimerize ethylene in 1‐butene (C4(α)=93 % (99+%)) at 30 bar C2H4 and 55 °C with AlEt3 as co‐catalyst, resulting in very high activity and selectivity for a molecular titanium catalyst (13000 g gTi−1 h−1, 93 % 1‐butene).

(Invited) In-Situ and Combinatorial Techniques for Spatial ALD

Atmospheric-pressure spatial atomic layer deposition (AP-SALD) and chemical vapor deposition (AP-CVD) have been developed in recent years as scalable techniques for the rapid deposition of oxide thin films on different substrates for a variety of applications. The atmospheric nature of these techniques facilitates the integration of characterization tools and the modification of the experimental setup to produce novel materials. Here we report in-situ electrical and optical characterization methods that have been developed for our AP-SALD/CVD system, as well as new techniques to deposit oxide films with nanoscale thickness gradients. The in-situ electrical measurements are enabled by a custom-designed, flexible printed circuit board substrate and the optical measurements are performed via reflectometry techniques. The thickness, resistance, and optical constants of prototypical AP-SALD films (ZnO and Al2O3) were monitored during depositions, providing insight into film nucleation and growth. The nanoscale thickness gradient films facilitate combinatorial high-throughput screening of devices, where a multitude of devices with varying film thicknesses can be fabricated on a single substrate. This combinatorial approach was applied to study the role of Al2O3 film thickness as an insulating layer in quantum-tunneling metal-insulator-metal diodes and as an encapsulation layer in metal halide perovskite solar cells.

A high-throughput drug combination screen identifies an anti-glioma synergism between TH588 and PI3K inhibitors

BackgroundGlioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor. More than half of GBMs contain mutation(s) of PTEN/PI3K/AKT, making inhibitors targeting the PI3K pathway very attractive for clinical investigation. However, so far, PI3K/AKT/mTOR inhibitors have not achieved satisfactory therapeutic effects in clinical trials of GBM. In this study, we aimed to develop a high-throughput screening method for high-throughput identification of potential targeted agents that synergize with PI3K inhibitors in GBM.MethodsA Sensitivity Index (SI)-based drug combination screening method was established to evaluate the interactions between BKM120, a pan-PI3K inhibitor, and compounds from a library of 606 target-selective inhibitors. Proliferation, colony and 3D spheroid formation assays, western blotting, comet assay, γ-H2AX staining were used to evaluate the anti-glioma effects of the top-ranked candidates. The drug combination effects were analyzed by the Chou-Talalay method.ResultsSix compounds were successfully identified from the drug screen, including three previously reported compounds that cause synergistic antitumor effects with PI3K/mTOR inhibitors. TH588, an putative MTH1 inhibitor exhibited significant synergy with BKM120 in suppressing the proliferation, colony formation and 3D spheroid formation of GBM cells. Further investigation revealed that both DNA damage and apoptosis were markedly enhanced upon combination treatment with TH588 and BKM120. Finally, activation of PI3K or overexpression of AKT compromised the anti-glioma efficacy of TH588.ConclusionsThe screening method developed in this study demonstrated its usefulness in the rapid identification of synergistic drug combinations of PI3K inhibitors and targeted agents.

A pharmacodynamic model of clinical synergy in multiple myeloma.

BackgroundMultiagent therapies, due to their ability to delay or overcome resistance, are a hallmark of treatment in multiple myeloma (MM). The growing number of therapeutic options in MM requires high-throughput combination screening tools to better allocate treatment, and facilitate personalized therapy.MethodsA second-order drug response model was employed to fit patient-specific ex vivo responses of 203 MM patients to single-agent models. A novel pharmacodynamic model, developed to account for two-way combination effects, was tested with 130 two-drug combinations. We have demonstrated that this model is sufficiently parameterized by single-agent and fixed-ratio combination responses, by validating model estimates with ex vivo combination responses for different concentration ratios, using a checkerboard assay. This new model reconciles ex vivo observations from both Loewe and BLISS synergy models, by accounting for the dimension of time, as opposed to focusing on arbitrary time-points or drug effect. Clinical outcomes of patients were simulated by coupling patient-specific drug combination models with pharmacokinetic data.FindingsCombination screening showed 1 in 5 combinations (21.43% by LD50, 18.42% by AUC) were synergistic ex vivo with statistical significance (P < 0.05), but clinical synergy was predicted for only 1 in 10 combinations (8.69%), which was attributed to the role of pharmacokinetics and dosing schedules.InterpretationThe proposed framework can inform clinical decisions from ex vivo observations, thus providing a path toward personalized therapy using combination regimens.FundingThis research was funded by the H. Lee Moffitt Cancer Center Physical Sciences in Oncology (PSOC) Grant (1U54CA193489-01A1) and by H. Lee Moffitt Cancer Center's Team Science Grant. This work has been supported in part by the PSOC Pilot Project Award (5U54CA193489-04), the Translational Research Core Facility at the H. Lee Moffitt Cancer Center & Research Institute, an NCI-designated Comprehensive Cancer Center (P30-CA076292), the Pentecost Family Foundation, and Miles for Moffitt Foundation.

Abstract 3523: A high-throughput screening strategy for the identification of novel lymphoproliferative elements

Abstract Introduction - Chimeric Antigen Receptor (CAR) T-cell therapy is effective against certain leukemias and lymphomas and shows promise for other incurable malignancies. Considerable challenges remain however to expand this platform technology beyond transplant-oriented hospital care. Centralized manufacturing of genetically modified T cells, lymphodepleting chemotherapy and patient management of current CAR-T therapies are associated with significant costs and treatment complexity. As a first step to reduce this treatment complexity, the present study describes a high throughput combinatorial domain library screening method to identify synthetic lymphoproliferative elements capable of driving in vivo expansion and survival of CAR-T cells in a lymphoreplete host without the homeostatic proliferation signals generated by lymphodepleting chemotherapy. Methods - High-diversity semi-rationally-designed combinatorial libraries of putative lymphoproliferative protein subdomains were DNA barcoded and assembled into a lentiviral vector co-expressing a ROR2-targeted CAR. Human PBMC were transduced with the library and cultured in vitro for several days. Purified cells were injected into mice bearing xenograft tumors modified to express the ROR2 antigen and compared to unmodified xenograft controls. The expansion rate of integrated cells was monitored weekly by quantitative PCR and, after 21 days of exposure, genomic DNA was isolated from blood, spleen and xenograft tumor tissues. Enriched barcodes were amplified using PCR and amplicons were subjected to HiSeq Next-Generation Sequencing. Barcode decoding was achieved using PacBio long read sequencing analysis to align full-length construct sequences with barcode quantitation. Results - Using this approach, putative CAR-T cell driver candidates and common key protein subdomains were identified that support selective in vivo expansion and survival of human lymphocytes in a tumor-bearing mouse model. Conclusion - Taken together, these results demonstrate that a high throughput combinatorial screening strategy with quantitative bioinformatics is a viable method for identifying protein domain combinations capable of selectively driving human CAR-T cells in vivo. These small synthetic combinatorial protein domains may facilitate lymphodepleting chemotherapy-free regimens and lower CAR-T cell doses in the future. Citation Format: Laurence Jadin, Hiba Shaban, Anirban Kundu, Gregory Schreiber, Scooter Willis, Farzad Haerizadeh, James Onuffer, Gregory Frost. A high-throughput screening strategy for the identification of novel lymphoproliferative elements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3523.

High-throughput combinatorial screening of multi-component electrolyte additives to improve the performance of Li metal secondary batteries

Data-driven material discovery has recently become popular in the field of next-generation secondary batteries. However, it is important to obtain large, high quality data sets to apply data-driven methods such as evolutionary algorithms or Bayesian optimization. Combinatorial high-throughput techniques are an effective approach to obtaining large data sets together with reliable quality. In the present study, we developed a combinatorial high-throughput system (HTS) with a throughput of 400 samples/day. The aim was to identify suitable combinations of additives to improve the performance of lithium metal electrodes for use in lithium batteries. Based on the high-throughput screening of 2002 samples, a specific combination of five additives was selected that drastically improved the coulombic efficiency (CE) of a lithium metal electrode. Importantly, the CE was remarkably decreased merely by removing one of these components, highlighting the synergistic basis of this mixture. The results of this study show that the HTS presented herein is a viable means of accelerating the discovery of ideal yet complex electrolytes with multiple components that are very difficult to identify via conventional bottom-up approach.

Synthesis of diN-Substituted Glycyl-Phenylalanine Derivatives by Using Ugi Four Component Reaction and Their Potential as Acetylcholinesterase Inhibitors

Ugi four component reaction (Ugi-4CR) isocyanide-based multicomponent reactions were used to synthesize diN-substituted glycyl-phenylalanine (diNsGF) derivatives. All of the synthesized compounds were characterized by spectroscopic and spectrometric techniques. In order to evaluate potential biological applications, the synthesized compounds were tested in computational models that predict the bioactivity of organic molecules by using only bi-dimensional molecular information. The diNsGF derivatives were predicted as cholinesterase inhibitors. Experimentally, all of the synthesized diNsGF derivatives showed moderate inhibitory activities against acetylcholinesterase (AChE) and poor activities against butyrylcholinesterase (BuChE). Compound 7a has significant activity and selectivity against AChE, which reveals that the diNsGF scaffold could be improved to reach novel candidates by combining other chemical components of the Ugi-4CR in a high-throughput combinatorial screening experiment. Molecular docking experiments of diNsGF derivatives inside AChE suggest that these compounds placed the phenylalanine group at the peripheral site of AChE. The orientations and chemical interactions of diNsGF derivatives were analyzed, and the changeable groups were identified for future exploration of novel candidates that could lead to the improvement of diNsGF derivative inhibitory activities.