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Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19. Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories. We developed a higher order consortium within the TBDA in which four pharmaceutical companies (GlaxoSmithKline, Sanofi, MSD, and Lilly) collectively collaborated with screeners at Weill Cornell Medicine, the Infectious Disease Research Institute (IDRI), and the National Institute of Allergy and Infectious Diseases (NIAID), pharmacologists at Rutgers University, and medicinal chemists at the University of North Carolina to screen ∼8900 β-lactams, predominantly cephalosporins, and characterize active compounds. In a striking contrast to historical expectation, 18% of β-lactams screened were active against Mtb, many without a β-lactamase inhibitor. One potent cephaloporin was active in Mtb-infected mice. The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

We identified an amino-benzothiazole scaffold from a whole cell screen against recombinantMycobacterium tuberculosisunder expressing the essential signal peptidase LepB. The seed molecule had two-fold higher activity against the LepB hypomorph. Through a combination of purchase and chemical synthesis we explored the structure activity relationship for this series; 34 analogs were tested for anti-tubercular activity and for cytotoxicity against eukaryotic cells. We identified molecules with improved potency and reduced cytotoxicity. However, molecules did not appear to target LepB directly and did not inhibit protein secretion. Key compounds showed good permeability, low protein binding, and lack of CYP inhibition, but metabolic stability was poor with short half-lives. The seed molecule showed good bactericidal activity against both replicating and non-replicating bacteria, as well as potency against intracellularM. tuberculosisin murine macrophages. Overall, the microbiological properties of the series are attractive if metabolic stability can be improved, and identification of the target could assist in development of this series.

The advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin-fixed paraffin-embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post-translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”. Here we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high-plex digital quantitation of proteins (147-plex) and RNA (whole transcriptome, > 18,000-plex) from a single FFPE sample. We demonstrated high concordance, R > 0.85, and minor change in sensitivity (2-fold higher protein expression levels of phospho-GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho-Thr231 Tau was >2-fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology. Citation Format: Shilah Bonnett, Alyssa Rosenbloom, Giang Ong, Mark Conner, Daniel Newhouse, Felicia New, Hiromi Sato, Chi Pha, Saskia Ilcisin, John Lyssand, Gary Geiss, Joseph M. Beechem. Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles. [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 4310.

The GeoMx࣪ Digital Spatial Profiler (DSP) enables high-plex, high-throughput spatial profiling and quantification from a single slide for either protein or RNA. To fully understand the interplay between RNA and protein in tissue, we have developed a novel multi analyte assay that allows for the profiling of both analytes from the area of interest (AOI) on a single slide. Here we describe the development and performance of the multi analyte assay on cell pellet array (CPA) and various tissues including tonsil and colorectal cancer (CRC) using a high plex GeoMx Protein Panel and the GeoMx Human Whole Transcriptome Atlas (WTA). We successfully identified conditions to detect RNA and protein simultaneously, with comparable sensitivity and specificity to the single analyte conditions. The data accurately matched cell lines in an unbiased correlation analysis between GeoMx WTA and the entire CCLE RNAseq database. Furthermore, the assay performance was maintained in tissue samples. Tonsil and colorectal cancer biopsies were profiled for RNA (whole transcriptome) and protein from the same AOI, allowing simultaneous measurements of transcriptional and translational regulation of key biological pathways. This multi-analyte profiling enables deeper characterization of precious biological samples that are available in limited quantities. Citation Format: Shilah Bonnett, Nicholas Confuorto, Kit Fuhrman, Giang Ong, Alyssa Rosenbloom, John Lyssand, Gary Geiss, Joseph Beechem. Multiomic analysis of whole transcriptome and high plex protein assays on a single FFPE slide [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 2029.

Background Understanding protein expression patterns within tissue compartments is imperative to investigating a range of biological questions. Historically, low plex immunohistochemical (IHC) approaches have been employed to assess the spatial heterogeneity of protein expression in tissue slices, but these techniques are of limited utility due to the challenge of measuring multiple targets in parallel. Compounding this limitation is the necessity of validating all antibodies which is resource intensive. Antibodies with poor quality have led to wasted time and resources, including false positives and non-reproducible results.1 2 Here we review the antibody validation process for the GeoMx® Digital Spatial Profiler (DSP) which enables investigation of high-plex, validated, spatially resolved protein targets from a single slide mounted formalin-fixed paraffin-embedded (FFPE) or fresh frozen sample. The robust validation process is in line with recent suggestions for antibody validation from SITC.3 Methods Unconjugated and oligo-conjugated antibodies are screened by IHC to assess staining sensitivity, patterns, and more importantly ensure that the oligo-conjugation has not adversely affected antibody performance. Upon approval by a pathologist, the antibodies are incorporated into a core or module and further validated using the GeoMx DSP. Using FFPE cell pellet arrays (CPAs) containing positive and negative control pellets, we assess the specificity as defined as a lack of signal in negative control pellets and a robust signal in positive control pellets. Antibodies with robust signals are then screened on tissue microarrays (TMAs) composed of healthy and diseased tissues to ensure that they will perform as expected in real samples and yield sufficient signal over background. Finally, after antibodies pass functional validation, we assess the performance of antibodies within panels of antibodies that will be commercialized. Results In total, approximately 60% of off-the-shelf antibodies tested for use in GeoMx assays pass the entire validation process and are put into commercial assays. Passing requirements include exhibiting a maximum positive signal divided by the limit of detection, plus two standard deviations (SD) that is greater than or equal to 5 in both CPAs and TMAs for individual antibodies; such a threshold gives a false positive rate of less than 10%. Conclusions Unvalidated or poorly validated antibodies can result in false positives and non-reproducible results. Following the robust validation process outlined here, approximately 40% of off-the-shelf antibodies are removed from panels, underscoring the importance of antibody validation prior to incorporating new antibodies into experiments. References Taussig MJ, Fonseca C, and Trimmer JS. Antibody validation: a view from the mountains. N Biotechnol. 2018; 45:1–8. Bordeaux J, Welsh AW, Agarwal S, Killiam E, Baquero MT, Hanna JA, Anagnostou VK, Rimm DL. Antibody validation. Biotechniques 2010;48(3):197–209. Taube, et al. The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation. J Immunother Cancer 2020;8(1):e000155.