Proprietary Library Research Articles

Findings from the individualized management of a patient with Acyl-CoA Oxidase-1 (ACOX1) deficiency: A bedside-to-bench-to-bedside strategy

Acyl-CoA Oxidase-1 (ACOX1) deficiency (MIM 264470) is an autosomal recessive disease characterized by impairments in the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, which is the first step in the catalysis of the β-oxidative breakdown of very long chain fatty acids (VLCFA) occuring in peroxisomes. The deleterious accumulation of VLCFA in several organs, including the brain, is a key biochemical feature of this disease which has devastating neurological consequences. ACOX1 deficiency is ultra-rare; as such, few studies have been conducted to determine the leading causes of symptoms or uncover new therapeutics. When confronted with one such case, we decided to bring drug discovery tools to the patient's bedside in an attempt to identify a cure. A skin biopsy was performed on a young patient with ACOX1 deficiency, following which screening technologies and mass spectrometry analysis techniques were applied to design a cellular assay that enabled the direct measurement of the effect of small molecules on the patient's primary fibroblasts. This approach is particularly well adapted to inherited metabolic disorders such as ACOX1 deficiency. Through the evaluation of a proprietary library of repurposable drugs, we found that the anthelmintic drug niclosamide led to a significant reduction in VLCFA in vitro. This drug was subsequently administered to the patient for more than six years. This study outlines the screening and drug selection processes. Additionally, we present our comprehensive clinical and biochemical findings that aided in understanding the patient's natural history and analysis of the progression of the patient's symptoms throughout the treatment period. Although the patient's overall lifespan was extended compared to the average age at death in severe early onset cases of ACOX1 deficiency, we did not observe any definitive evidence of clinical or biochemical improvement during niclosamide treatment. Nonetheless, our study shows a good safety profile of long-term niclosamide administration in a child with a rare neurodegenerative disease, and illustrates the potential of individualized therapeutic strategies in the management of inherited metabolic disorders, which could benefit both patients and the broader scientific and medical communities.

Trueness and precision of combined healing abutment scan body system scans at different sites of maxilla after multiple repositioning of the scan body

ObjectivesTo evaluate the accuracy of the scans of the combined healing abutment-scan body (CHA-SB) system located at different sites of the maxilla when SBs are replaced in between each scan. MethodsThree SBs were seated into HAs located at the central incisor, first premolar, and first molar sites of a maxillary model inside a phantom head, and the model was scanned extraorally (CEREC Primescan SW 5.2). This procedure was repeated with new SBs until a total of 10 scans were performed. Standard tessellation language files of CHA-SBs at each implant location were isolated, transferred into analysis software (Geomagic Control X), and superimposed over the proprietary library files to analyze surface (root mean square), linear, and angular deviations. Trueness and precision were evaluated with one-way analysis of variance and Tukey tests. The correlation between surface and angular deviations was analyzed with Pearson's correlation (α=0.05). ResultsMolar implant scans had the highest surface and angular deviations (P≤.006), while central incisor implant scans had higher precision (surface deviations) than premolar implant scans (P=.041). Premolar implant scans had higher accuracy than central incisor implant scans on the y-axis (P≤.029). Central incisor implant scans had the highest accuracy on the z-axis (P≤.018). A strong positive correlation was observed between surface and angular deviations (r = 0.864, P<.001). ConclusionCentral incisor implant scans mostly had high accuracy and molar implant scans mostly had lower trueness. SBs were mostly positioned apically; however, the effect of SB replacement can be considered small as measured deviations were similar to those in previous studies and the precision of scans was high. Clinical SignificanceRepositioning of scan bodies into healing abutments would be expected to result in similar single crown positioning regardless of the location of the implant, considering high scan precision with the healing abutment-scan body system. The duration of the chairside adjustments of crowns in the posterior maxilla may be longer than those in the anterior region.

Telerehabilitation with ARC Intellicare to Cope with Motor and Respiratory Disabilities: Results about the Process, Usability, and Clinical Effect of the "Ricominciare" Pilot Study.

"Ricominciare" is a single-center, prospective, pre-/post-intervention pilot study aimed at verifying the feasibility and safety of the ARC Intellicare (ARC) system (an artificial intelligence-powered and inertial motion unit-based mobile platform) in the home rehabilitation of people with disabilities due to respiratory or neurological diseases. People with Parkinson's disease (pwPD) or post-COVID-19 condition (COV19) and an indication for exercise or home rehabilitation to optimize motor and respiratory function were enrolled. They underwent training for ARC usage and received an ARC unit to be used independently at home for 4 weeks, for 45 min 5 days/week sessions of respiratory and motor patient-tailored rehabilitation. ARC allows for exercise monitoring thanks to data from five IMU sensors, processed by an AI proprietary library to provide (i) patients with real-time feedback and (ii) therapists with information on patient adherence to the prescribed therapy. Usability (System Usability Scale, SUS), adherence, and adverse events were primary study outcomes. Modified Barthel Index (mBI), Barthel Dyspnea Index (BaDI), 2-Minute Walking Test (2MWT), Brief Fatigue Inventory (BFI), Beck Depression or Anxiety Inventory (BDI, BAI), and quality of life (EQ-5D) were also monitored pre- and post-treatment. A total of 21 out of 23 eligible patients were enrolled and completed the study: 11 COV19 and 10 pwPD. The mean total SUS score was 77/100. The median patients' adherence to exercise prescriptions was 80%. Clinical outcome measures (BaDI, 2MWT distance, BFI; BAI, BDI, and EQ-5D) improved significantly; no side effects were reported. ARC is usable and safe for home rehabilitation. Preliminary data suggest promising results on the effectiveness in subjects with post-COVID condition or Parkinson's disease.

A study on the implementation of Koha cataloguing module in Malaysian academic libraries

The study explores the experiences of the Malaysian academic libraries’ cataloguers in their use of the cataloguing module of Koha Open Source Software (OSS). The purpose of this study is to shed light on issues concerning Koha cataloguing module and the local practices in policy and procedure while utilising the module. An exploratory sequential research design was adopted for the study involving an online focus group interview and a questionnaire survey as the data collection techniques. Twenty-two academic libraries from public and private universities in Malaysia were purposively sampled. A total of six academic libraries participated in the focus group, whereas 41 participants took part in the survey. The study highlighted academic libraries in Malaysia embrace Koha for two main reasons - popularity as and OSS and budget constraints for a proprietary library system. The findings of the study revealed that missing records is a significant issue that cataloguers will have to deal with and there is a lack of certain features available in the Koha cataloging module, such as the label creator and search feature. Apart from that, using Koha for cataloguing does not affect the cataloguing practices in terms of policy, but it does affect the cataloguing procedure. Satisfaction level with Koha cataloguing module is high. Other academic libraries in Malaysia that have not yet made a move to Koha or are in the midst of doing so would benefit from this study.

A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.

Abstract 2946: Depleting hCCR8 mAb Therapy #1: Characterization of a broad collection of anti-hCCR8 mAbs

Abstract Background: Tumor-infiltrating Tregs (TITR) constitute a sub-family of immuno-suppressive cells abundantly found in multiple solid cancers. They play a critical role in tumor progression and their specific depletion has been recently proposed as a novel therapeutic strategy to fight cancers. Based on the transcriptional analysis of tumor infiltrating immune cells, the G-protein coupled receptor CCR8 was found to be expressed on murine and human TITR, but not on proinflammatory effector T cells. The CCR8 receptor therefore represents a unique TITR target for the development of novel depletive antibody-based therapeutics. Methods: We have generated a broad collection of monoclonal antibodies (mAb) directed against the human CCR8 receptor. This collection was widely characterized using different sources of recombinant and native hCCR8 positive cell subsets. Moreover, their binding and activities in several functional assays were assessed with different approaches (BRET, Flow Cytometry, including the inhibition of recruitment of different G-proteins to the CCR8 receptor). A comprehensive molecular and pharmacological characterization of each mAb of our proprietary library was generated. mAb binding against different peptides mimicking different states of post-translational modifications of the N-terminus of CCR8 was also monitored. Finally, using two surrogate antibodies directed against the mouse receptor, we tested the role of CCR8 antagonism and Treg depletion in multiple syngeneic models. Results: A comprehensive characterization of Domain Therapeutics’ mAb library revealed distinct mAb cellular reactivity profiles, different epitope recognition patterns (based on binding to different peptides with different post-translational modifications), subnanomolar antagonist activity of downstream signaling, and insurmountable property with regards to the CCR8 ligand CCL1. Moreover, we demonstrated that treatment with a depleting anti-mCCR8 mAb translated into robust anti-tumor activity in multiple syngeneic mouse models. Conclusion: Due to its high and relatively specific expression on tumor-infiltrating Tregs, CCR8 represents an attractive novel target to derive novel immunotherapies. At Domain Therapeutics, a mAb library of several dozen antibodies with distinct and differentiated binding and pharmacological activity profile was successfully discovered and patent-protected. This collection constitutes a unique source of future clinical candidates for the development of a best-in-class well-differentiated anti-hCCR8 depleting antibody for the treatment of cancers. Citation Format: Claudine Vermot-Desroches, Iseulys Richert, Malaury Schappler, Alice Gentil Dit Maurin, Megane Jeannelle, Solene Rose, Luc Baron, Quentin Ruet, Camille Dietsch, Pauline Urquia, Safia Ayachi, Orphee Blanchard, Maria Dolores Garcia Fernandez, Christel Franchet, Nathalie Lenne, Stephan Schann. Depleting hCCR8 mAb Therapy #1: Characterization of a broad collection of anti-hCCR8 mAbs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2946.

Abstract 3471: A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment

Abstract Tumor-associated macrophages (TAMs) are important players to maintain immunosuppression within the tumor microenvironment (TME) and attenuate the function of effector immune cells to promote tumor survival. High density of TAMs is well-recognized as a feature of tumor progression and poor prognostic factor across various tumor types. Therefore, therapeutic strategies to dampen TAMs’ suppressive potential in TME and reprogram them towards a pro-inflammatory phenotype are being increasingly appreciated as being paramount for an effective immunotherapy. Nevertheless, the key mediators of TAM-induced immunosuppression remain largely unknown, identification of which can lead to the development of novel immunotherapeutic strategies aimed at re-educating the immunosuppressive TAMs to an anti-tumor phenotype. To systematically interrogate the genes responsible for the immune-suppressive phenotype of TAMs, we developed a functional, high-throughput genetic screening platform (iOTarg™) based on human primary monocyte-derived M2-like macrophages. Using CRISPR, we knocked out a proprietary library of well-expressed and druggable genes (1400 genes) in two independent donor-derived M2-like macrophages and cocultured them with activated autologous effector T cells (TCs). We achieved nearly complete knockout (KO) efficacy over a range of control genes. Functional impact of individual KO on macrophage viability and phenotype, as well as on TC activity, was measured using multiparametric assay readouts. Initial hits were reconfirmed in three additional donors in a secondary screen that integrated an additional tumor lysis readout. As expected, knockout of CSF1R, which is essential for macrophage maintenance, resulted in a dramatic loss of macrophage cell viability, whereas KO of established TAM markers, TREM2 and Clever-1, induced a change in macrophage phenotype. Furthermore, KO of immune-inhibitory receptor LILRB2 reduced the M2-like phenotype and restored TC activity. Inhibition of M2 activation of macrophages and subsequent increase in TC activation culminated in strong tumor cell killing for a subset of genes, highlighting an untapped repertoire of novel TAM-associated immune-checkpoint targets. Taken together, we report for the first time a highly sophisticated target discovery platform that addresses the functional role of any TAM-expressed gene in regulating macrophage viability, phenotype, T cell activity and even its gross impact on tumor cell lysis, all done in a high-throughput format employing CRISPR-edited primary human immune cells and multi-parametric functional immunological assays. As a result, we could confirm well-known targets in clinical testing, as well as identify additional novel targets that could lead to first-in-class, TME-based therapeutics and expansion of treatment options in immune oncology. Citation Format: Kritika Sudan, Tillmann Michels, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Lucille Albert, Valentina Volpin, Adriana Turqueti-Neves, Ronny Milde, Nisit Khandelwal. A function-based high-throughput discovery platform, myeloid iOTarg, identifies novel immune checkpoints of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3471.

Abstract 481: Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma

Abstract High-risk neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Despite extensive study, treatments remain highly toxic and are often insufficient to cure. Thus, there is an urgent need to develop more effective and less toxic treatments for children with NB. NB commonly displays reliance on a relatively small cohort of transcription factors (TFs) and epigenetic proteins to drive cell growth, however, many of these targets are inaccessible with conventional small molecules. In recent years, new modalities such as targeted protein degradation (TPD) have emerged. TPD may permit small molecule degradation of traditionally “undruggable” targets, such as TFs. This makes TPD a particularly attractive modality for defining new therapeutic strategies for high-risk NB. One category of TPD agents are molecular glues (MGs). MGs are monovalent small molecules capable of simultaneously binding to an E3 ligase receptor such as CRBN (cereblon), and a target protein of interest. Importantly, in comparison to conventional protein inhibitors, MGs do not require a conventional binding pocket on the protein of interest to bind to it, and can be utilized in sub-stoichiometric concentrations. Thus, this may result in the recruitment, polyubiquitination, and subsequent proteasomal degradation of proteins previously thought to be “undruggable.” Here, we demonstrate that high-risk NB cells are critically sensitive to a cluster of previously unknown MGs. We performed high-throughput screening of a proprietary library (&amp;gt;4000 unique compounds) of MGs in two MYCN-amplified NB cell lines, Kelly and MHHNB11. These two cell lines demonstrate a strikingly close correlation in Cell-Titer Glo-based growth response to MGs, as compared with an outgroup of other MG-responsive cancer cell lines. We identified a unique subcluster of 112 common MGs that display &amp;gt;70% growth inhibition in both cell lines. To identify the mechanism of cell growth inhibition, we established Kelly cells with stable knockout of CRBN, and coupled these with screening to confirm that the majority of these MGs (84%; 94/112) require CRBN expression. To rule out common off-target effects of MGs, candidate compounds were then tested in cells transgenically expressing either wild-type or degradation-resistant GSPT1 (G575N) constructs, revealing a subset of MGs retaining cytotoxicity without effecting GSPT1 protein levels. Current studies are focused on determining the targets of these high confidence MGs, as a foundation for preclinical in vivo studies. These studies will identify new compounds with defined mechanisms of action, with an ultimate goal of developing new treatments that are more effective and less toxic for children with NB. Citation Format: Ian Delahunty, Gisele Nishiguchi, Yang Zhang, Abigail Fish, Anand Mayasundari, Jun Qi, Brian Abraham, Anang Shelat, Zoran Rankovic, Adam Durbin. Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 481.

Abstract 3377: Liquid biopsy analysis reveals orthologs of actionable human cancer variants in dogs

Abstract Genotype-matched therapeutics have become widely adopted in the management of human cancers and can be guided by testing tumor tissue or cell-free DNA from plasma. OncoKB (Oncology Knowledge Base) is the first FDA-recognized database that contains information about these targetable somatic variants and corresponding therapeutic agents, including levels of evidence. Many of the human OncoKB variants have orthologs in the canine genome. This study was conducted to determine the feasibility of detecting these orthologs in dogs using liquid biopsy. This study involved two cohorts: The first was a cohort of 619 client-owned dogs with a variety of cancer diagnoses across all disease stages enrolled in a clinical validation study for a liquid biopsy test (herein the “research cohort”). All dogs had pre-treatment plasma samples collected at enrollment, and a subset of dogs also had matched tumor tissue collected. The second cohort comprised 457 client-owned dogs that had testing as part of the clinical deployment of the above-mentioned liquid biopsy test (herein the “clinical cohort”); 31% of these dogs had samples submitted as an aid in diagnosis (for dogs in which cancer was suspected clinically), and 69% had samples submitted for screening (in dogs with no a priori suspicion of cancer). Blood samples (and tumor samples, when available) were subjected to DNA extraction, proprietary library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. Only canine orthologs of variants in the OncoKB database were evaluated in this study. Variants were called if they were observed in plasma at an allele frequency of at least 0.5%, with at least 6 unique supporting reads. Variants identified from plasma were subsequently genotyped in tissue without additional variant-level filtering. Analysis of plasma identified canine orthologs of OncoKB variants in ~8% of dogs in the research cohort, and ~4% of dogs in the clinical cohort. The higher percentage observed in the research cohort is likely due to the higher percentage of patients with confirmed cancer diagnosis. In the patients with an OncoKB ortholog in the research cohort, the dog had a cancer type that matched the human indication for the detected variant in 15% of cases. Of dogs in which an ortholog was identified in plasma, 44% had matched tumor tissue; and the variant observed in plasma was confirmed in tissue in &amp;gt;50% of these subjects. This study demonstrates the feasibility of detecting canine orthologs of targetable human cancer somatic variants in the blood of dogs using next-generation sequencing. Although it is currently unclear how human databases can be used to inform targeted treatment decisions in dogs, these findings may have relevance for comparative oncology studies and future precision therapeutics development for both species. Citation Format: Daniel S. Grosu, Ilya Chorny, Kristina M. Kruglyak, John A. Tynan, Susan C. Hicks, Todd A. Cohen, Allison L. O'Kell, Jill M. Rafalko, Jason Chibuk, Angela L. McCleary-Wheeler, Andi Flory, Dana W. Tsui. Liquid biopsy analysis reveals orthologs of actionable human cancer variants in dogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3377.

Open-Source and Proprietary Library Automation Software: A Comparative Academic Librarian's Perspective

The purpose of this study was to get an understanding of how academic librarians perceive open-source and proprietary library software. The researchers used a survey research approach and a self-constructed questionnaire as data collection tool. The study targeted 103 library professionals working at HEC-recognized degree awarding institutions (DAIs) and universities in Lahore, both public and private sectors, yielding an 84% response rate. To meet research objectives, descriptive and inferential statistics were applied. Study findings explored that library software should have the facility of advanced searching, library standards, and a user-friendly interface. Comparative analysis revealed that open-source library software contains more modules and requires highly skilled employees. On the other hand, human resources with minor skills can use proprietary software. Factors that prevent adopting OSS for academic libraries include a lack of technical support, skilled and motivated library professionals, inadequate cooperation between supervisors and subordinates, and insufficient training opportunities. Experienced library professionals should guide and offer full cooperation in the selection and implementation of software. Library schools and associations should play a decisive role through school curriculum and professional development programs in preparing graduates and practitioners for embracing modern technologies. Open-source software developers should also organize training programs for software users to work independently in libraries.

Activity of 1-aryl-4-(naphthalimidoalkyl) piperazine derivatives against Leishmania major and Leishmania mexicana.

A series of 1-aryl-4-(phthalimidoalkyl) piperazines and 1-aryl-4-(naphthalimidoalkyl) piperazines were retrieved from a proprietary library based on their high structural similarity to haloperidol, an antipsychotic with antiparasitic activity, and assessed as potential antileishmanial scaffolds. Selected compounds were tested for antileishmanial activity against promastigotes of Leishmania major and Leishmania mexicana in dose-response assays. Two of the 1-aryl-4-(naphthalimidoalkyl) piperazines (compounds 10 and 11) were active against promastigotes of both Leishmania species without being toxic to human fibroblasts. Their activity was found to correlate with the length of their alkyl chains. Further analyses showed that compound 11 was also active against intracellular amastigotes of both Leishmania species. In promastigotes of both Leishmania species, compound 11 induced collapse of the mitochondrial electrochemical potential and increased the intracellular Ca2+ concentration. Therefore, it may serve as a promising lead compound for the development of novel antiparasitic drugs.

Abstract 1398: Structure-based design of small macrocyclic CDK9 degraders as chemical biology tools and beyond

Abstract Small molecule macrocyclic kinase inhibitors have attracted significant attention in drug discovery over the past years with drugs approval such as lorlatinib demonstrating the clinical relevance of this approach. We developed our expertise to further optimize those cyclic molecules. Having low molecular weight, they favorably alter the biological and physiochemical properties as well as selectivity, as compared to their linear parent, yielding high-quality drug candidates. While focused on kinase inhibitors, macrocyclic derivatives could be potentially turned into bifunctional protein degrader molecules useful for selective cellular knockdown of targeted proteins and investigation of the pharmacological effects. With macrocyclic “probes” from our proprietary library in nanomolar range IC50, we turned our attention to CDK9 inhibitors with good selectivity profile against CDK1/2/5/7. Previous CDK9 degraders based on acyclic inhibitors have used thalidomide as recruiter of the CRL4CRBN, resulting in successful ubiquitination and proteasomal degradation of the protein of interest. Applying a three-step approach, we first defined the vector of substitution on our macrocyclic “probes” by homology modeling with known acyclic ligands/CDK9 co-crystal structures. Having defined 2 substitution patterns, we synthesized and tested in a biochemical assay the novels “probes” vectorized with the pro-linker moieties, without loss of activity. ODS’7152 was further derived into fully bifunctional molecules with variation on the linker’s nature and length, keeping thalidomide as the E3 ligase recruiter. Biophysical and biological properties were further evaluated in an MTS assay against several cell lines (HCT116, Jurkat E6.2, MOLT-4 and MV4-11) to assess cell proliferation and viability. The E3 Ligase engagement along with cell penetration was measured in a NanoBRET™ assay and compared to MDR_MDCK permeability. Finally, a selection of 4 PROTACs were evaluated in a dose response assay on MV4-11 for protein degradation phosphorylation of CTD-pol II and selectivity among several CDKs. Our findings support the rapid derivatization of macrocyclic “probes” inhibitors into macrocyclic-PROTAC as a strategy to generate early chemical biology tools with maintained potency and selectivity between homologous targets. Optimization of the physico-chemical and ADME properties of molecules can lead to drug candidates with distinct pharmacological effects as compared to the parent linear kinase inhibitors. Citation Format: Nerina Dodic, Nicolas Ancellin, Morgane Bordessoules, Anne Bouillot, Nathan Butin, Cedric Charrier, Marie-Helene Fouchet, Alain Laroze, Anne-Pascale Luzy, Alexandre Moquette, Julia Pilot, Guillaume Serin, Jean-François Mirjolet, Fabrice Viviani, Christophe Parsy. Structure-based design of small macrocyclic CDK9 degraders as chemical biology tools and beyond [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 1398.

Abstract 5456: Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations

Abstract FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), and its activating mutations including internal tandem duplications (ITDs) and missense point mutations in the tyrosine kinase domain (TKD) are found in approximately 30% of AML patients. Although the patients initially well responded to FLT3 inhibitors such as gilteritinib, the most cases relapsed within a few months after the initiation of treatment. For these patients who relapsed after FLT3 inhibitor-based therapy, no effective treatment is available. Mechanisms of resistance are not fully elucidated, but FLT3-ITD-TKD double mutant is considered as one of possible mechanisms. We have discovered a novel FLT3 inhibitor OTS447 that shows the potent and selective inhibition in both FLT3-ITD and FLT3-ITD-TKD double mutants. OTS447 was identified through the screening of our in-house proprietary compound library. It showed potent inhibitory activity against FLT3 with an IC50 value of 0.19 nM. The selectivity of OTS447 was investigated by human kinase profile assay. Among 371 human kinases tested, there were only seven (including FLT3) whose activity were inhibited by 80% or more at 5 nM of OTS447. We then examined the anti-proliferative effects in FLT3-ITD and -wild type (WT) AML cell lines. The proliferation of FLT3-ITD cell lines, MV4-11 and MOLM13, was more strongly suppressed than that of FLT3-WT cell lines. We also investigated the inhibitory effects to FLT3-ITD-TKD double mutants using Ba/F3 cells. FLT3-ITD-D835Y mutant was inhibited as strongly as FLT3-ITD mutant, and FLT3-ITD-F691I mutant was more strongly inhibited than Ba/F3 parental cells. These data suggest that OTS447 has selective inhibitory activity against FLT3 mutants. To confirm that FLT3 inhibition by OTS447 leads to the anti-proliferative effect, FLT3 autophosphorylation and phosphorylation of downstream molecules such as STAT5, ERK and AKT were examined. Both autophosphorylation of FLT3 and phosphorylation of downstream molecules were decreased by OTS447 in dose-dependent manner. Moreover, treatment of OTS447 induced the apoptosis and increased sub-G1 population in MV4-11 cell. Finally, we tested OTS447 anti-tumor effect using MV4-11 mouse xenograft model. OTS447 showed potent growth inhibition against MV4-11 tumor in dose-dependent manner. In summary, we discovered OTS447, a potent and selective FLT3 inhibitor, that can inhibit not only ITD mutation but also ITD-TKD mutations of the FLT3 gene. OTS447 possesses cytotoxic activity induced by inhibition of FLT3 signaling pathway and has anti-tumor activity in mouse xenograft model. We are pursuing further optimization of the inhibitor aiming for the effective treatment of AML patients with several types of FLT3-activating mutations. Citation Format: Yasuhide Okamoto, Naofumi Takamatsu, Takashi Miyamoto, Akiko Taira, Kozue Toyota, Yukiho Imai, Shinobu Fujii, Rumiko Ono, Kyoko Adachi, Yo Matsuo, Suyoun Chung, Jae-Hyun Park. Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations [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 5456.

Discovery of 2-Phenylquinolines with Broad-Spectrum Anti-coronavirus Activity.

A selection of compounds from a proprietary library, based on chemical diversity and various biological activities, was evaluated as potential inhibitors of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a phenotypic-based screening assay. A compound based on a 2-phenylquinoline scaffold emerged as the most promising hit, with EC50 and CC50 values of 6 and 18 μM, respectively. The subsequent selection of additional analogues, along with the synthesis of ad hoc derivatives, led to compounds that maintained low μM activity as inhibitors of SARS-CoV-2 replication and lacked cytotoxicity at 100 μM. In addition, the most promising congeners also show pronounced antiviral activity against the human coronaviruses HCoV-229E and HCoV-OC43, with EC50 values ranging from 0.2 to 9.4 μM. The presence of a 6,7-dimethoxytetrahydroisoquinoline group at the C-4 position of the 2-phenylquinoline core gave compound 6g that showed potent activity against SARS-CoV-2 helicase (nsp13), a highly conserved enzyme, highlighting a potentiality against emerging HCoVs outbreaks.

Congruence between the meshes of a combined healing abutment-scan body system acquired with four different intraoral scanners and the corresponding library file: An in vitro analysis

ObjectivesTo investigate the congruence between the meshes of a combined healing abutment-scan body (CHA-SB) system acquired with four different intraoral scanners and the corresponding library file. Material and methodsA CHA-SB was fixed to an implant at the right first molar position in a dentate mandibular model and digitized by using 4 different intraoral scanners (IOSs) [TRIOS 3 (T3), Omnicam (OC), Primescan (PS), and Virtuo Vivo (VV)] (n = 8) and an industrial grade optical scanner (ATOS Core 80) (n = 1) to generate standard tessellation language (STL) files of the test scans (CHA-SB-STLs) and the master reference model scan (MRM-STL). A reverse engineering software (Studio Geomagic X) was used to superimpose the proprietary library file of the CHA-SB over the generated STL files. Root mean square (RMS) values representing the deviations between the library file and the superimposed STL files were statistically analyzed by using 1-way ANOVA (α=0.05). Qualitative analysis of the deviations was performed by visual inspection. ResultsDifferences between the congruence of the library file and the CHA-SB scans among different IOSs were nonsignificant (F = 1.619, df= 3, P = .207). The single best result was 29 ± 28.9 µm for OC, 30.8 ± 29.6 µm for VV, 35.6 ± 35.5 µm for T3, and 39.5 ± 39.2 µm for PS, which were all above the deviation value of the scan performed by using the industrial-grade scanner (23.2 ± 23.2 µm). ConclusionThe dimensional congruence between the library file and the STL file of the CHA-SB system scans was similar when intraoral scanners with different acquisition technologies were used to scan a model with an implant. Clinical significanceScans of the tested intraoral scanners may result in crowns with similar positional accuracy, given the similarities in congruence of their scans with the library file.

Abstract P221: Discovery of a covalent inhibitor for an oncogenic mutant RhoAY42C

Abstract Ras homolog family member A (RhoA) is a member of a larger family of Rho proteins of small GTPases, with structural and signaling similarities to Ras proteins. RhoA is involved in multiple cellular processes including cytoskeleton regulation and actin-myosin contractility, etc. Recent studies have shown that the most common RhoA mutation in diffuse gastric cancer (DGC), Y42C, causes a gain-of-function and is associated with the loss of the E-cadherin gene CDH (CDH1). Inhibitors of RhoAY42C could be promising agents for treating DGC that harbors CDH1 mutation/deletion. BridGene expressed RhoAY42C in cells, screened our proprietary covalent library against the RhoAY42C live cells, and employed our chemoproteomic platform IMTACTM (Isobaric Mass Tagged Affinity Characterization) to detect covalent labeling of the mutant RhoA protein. A pilot screening led to the successful identification of a covalent ligand of RhoAY42C, BGS1933. Mass spec analysis has confirmed the covalent modification of Cys42 in RhoA by BGS1933. Dose-dependent binding of BGS1933 to RhoAY42C was measured using an orthogonal fluorescence-based assay. The Cancer Genome Atlas (TCGA) study shows that ~1% DGC patients harbor the oncogenic mutant RhoAY42C. The discovery of a covalent inhibitor of RhoAY42C will enable us to validate the role of RhoAY42C in DGC and facilitate the development of new first-in-class targeted therapies for the treatment of gastric cancer. Citation Format: Shirley Guo, Ping Cao. Discovery of a covalent inhibitor for an oncogenic mutant RhoAY42C [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P221.

Abstract LBA004: Identification of GSPT1-directed molecular glue degrader (MGD) for the treatment of Myc-driven breast cancer

Abstract The Myc family of transcription factors is a well-established driver of human cancers. However, despite being amongst the most frequently mutated, translocated and overexpressed oncogenes, no therapy directly targeting the Myc family members has been developed to date. Abnormal activation of Myc results in uncontrolled cell growth that is associated with high translational output and ramp up of the protein translational machinery. This creates a dependency to protein translation and in turn represents a potential therapeutic vulnerability for Myc-driven tumors. Based on these considerations, we hypothesized that targeting the translational termination factor GSPT1, a key player of protein synthesis, may constitute a vulnerability for Myc-driven tumors. Using our proprietary Quantitative and Engineered Elimination of Neosubstrates (QuEENTM) platform we characterized and explored the known G-loop degron in GSPT1 that renders it amenable to cereblon-induced degradation by molecular glue degraders (MGDs). We rationally designed and subsequently screened a proprietary library of cereblon-binding small molecules, including GSPT1-directed MGDs, in human mammary epithelial cells (HMECs) expressing doxycycline-inducible c-Myc. Doxycycline treatment led to sustained c-Myc expression and as a consequence to the induction of key biomarkers of enhanced protein translation, such as phospho 4EBP1 (p4EBP1). We identified MRT-048 as a potent and highly selective GSPT1 degrader and demonstrated its ability to induce cell death in Myc-driven HMEC cells whilst sparing control cells (EC50 0.64 μM vs 30 μM respectively). This confirmed the selective vulnerability of Myc-driven cell growth to GSPT1 degradation. In follow-up studies, we confirmed the correlation between p4EBP1 as biomarker of Myc-activation and sensitivity to MRT-048 in a large panel of breast cancer cell lines. Moreover, MRT-048 treatment of animals xenografted with breast cancer cells induced tumor regression and was associated with complete GSPT1 degradation. Mechanistically, we observed that GSPT1 degradation induced by MRT-048 led to inhibition of genes regulated by Myc and ribosomal stalling at stop codons of several mRNAs. Additionally, polysome profiling of cancer cells treated with MRT-048 was associated with a global reduction of the intensities of the polysome peaks and concomitant increase in the monosome peaks as previously observed in GSPT1 knockdown experiments, suggesting that GSPT1 degradation by our MGD molecules affects both the termination and initiation stages of protein translation. We believe these data support the therapeutic potential of GSPT1-directed MGDs in Myc-driven tumors dependent on protein translation machinery. Citation Format: Gerald Gavory, Bernhard Fasching, Debora Bonenfant, Amine Sadok, Ambika Singh, Martin Schillo, Vittoria Massafra, Anne-Cecile d’Alessandro, John Castle, Mahmoud Ghandi, Agustin Chicas, Frederic Delobel, Alexander Flohr, Giorgio Ottaviani, Thomas Ryckmans, Anne-Laure Laine, Oliv Eidam, Hannah Wang, Ilona Bernett, Laura Chan, Chiara Gorrini, Theo Roumiliotis, Jyoti Choudhary, Yann-Vai LeBihan, Marc Cabry, Mark Stubbs, Rosemary Burke, Rob Van Montfort, John Caldwell, Rajesh Chopra, Ian Collins, Silvia Buonamici. Identification of GSPT1-directed molecular glue degrader (MGD) for the treatment of Myc-driven breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA004.

‘Very useful as well as ornamental’: social libraries in early American communities

Responding to previous analyses of eighteenth-century subscription libraries which focus primarily on the ‘useful’ — namely, access to books — this article instead considers the ‘ornamental’ aspects of library membership. Taking the colony of New York as a case study, it explores the ways in which the members of disparate, individual proprietary libraries were active participants in a broader, Atlantic-wide social movement, balancing elements of politeness, sociability, and an emerging civic society with the practical quest for the acquisition of knowledge. Primarily comprising an analysis of the early records of the New York Society Library, this article forms a prosopographical study of the library’s membership before the American Revolution, before exploring the social, commercial and familial connections between members. It ultimately concludes that early American social libraries both shaped and were shaped by the communities which they served, and highlights the potential of biographical approaches to library history for enhancing current understandings of early American communities.

Validation of an ASIC1a Ligand-Gated Assay on an Automated Patch Clamp Platform and its Use for Novel Ligand Screening

There is growing interest in automated patch clamp assays for ligand-gated targets, many of which are expressed throughout the nervous system and underlie complex neurological diseases. The Acid-Sensing Ion Channel (ASIC) family comprises combinations of ASIC1-4 proteins that form cation-selective channels in the central and peripheral nervous system. ASIC1a proteins are involved in synaptic plasticity, and implicated in stroke, ischemia and pain. Small molecules and toxins modulate ASIC1a channels, and fragments can bind to ASIC3 channels (Wolkenberg et al., 2010), so we screened a diverse low molecular weight library for novel ASIC1a modulators. All data was generated on the gigaseal QPatch48 system (Sophion) using coated metal pipetting tips and a CHO cell line stably expressing human ASIC1a. Currents were elicited from a holding potential of −60 mV by rapid application of saline or compound-containing solutions of varied pH. EC50 of pH activation and IC50 of test compound inhibition were determined by cumulative applications to the same cell. A proprietary low MW library was tested at 200 μM (N=2) and active hits were confirmed by cumulative IC50 testing (N=3). We validated the pharmacology of the hASIC1a cell line using toxins (e.g. IC50 of 120 nM for Mambalgin-3), small molecules (Benzamil IC50 of 4.0 μM), and clinical development compounds, which paved the way to screen a proprietary library of low molecular weight compounds in an effort to find chemical building blocks for the design of novel and efficacious ASIC1a modulators. A hit rate of 3.0% was achieved which included several novel scaffolds with low μM potency, which can be used in biophysical binding assays and structure-based drug design to develop ligand-efficient modulators of the human ASIC1a channel.

A Novel N-Substituted Valine Derivative with Unique Peroxisome Proliferator-Activated Receptor γ Binding Properties and Biological Activities.

A proprietary library of novel N-aryl-substituted amino acid derivatives bearing a hydroxamate head group allowed the identification of compound 3a that possesses weak proadipogenic and peroxisome proliferator-activated receptor γ (PPARγ) activating properties. The systematic optimization of 3a, in order to improve its PPARγ agonist activity, led to the synthesis of compound 7j (N-aryl-substituted valine derivative) that possesses dual PPARγ/PPARα agonistic activity. Structural and kinetic analyses reveal that 7j occupies the typical ligand binding domain of the PPARγ agonists with, however, a unique high-affinity binding mode. Furthermore, 7j is highly effective in preventing cyclin-dependent kinase 5-mediated phosphorylation of PPARγ serine 273. Although less proadipogenic than rosiglitazone, 7j significantly increases adipocyte insulin-stimulated glucose uptake and efficiently promotes white-to-brown adipocyte conversion. In addition, 7j prevents oleic acid-induced lipid accumulation in hepatoma cells. The unique biochemical properties and biological activities of compound 7j suggest that it would be a promising candidate for the development of compounds to reduce insulin resistance, obesity, and nonalcoholic fatty liver disease.