Accelerate Drug Discovery With Small Molecule Microarrays

Small Molecule Microarray (SMM) as a tool for rapid, efficient hit-identification against both protein and RNA targets A D A M B U C K L E , J U L I A U N S I C K E R , T A N I A H A N C O C K , R I C H A R D C H E N Small Molecule Microarray (SMM) is an established screening technique for identifying drug-like Small Molecule (SM) hits against a variety of target types. In recent years, SMM has enjoyed renewed interest from drug discovery groups due to its efficiency and speed, its status as a distinct and complementary option to other hit discovery methods, and its compatibility with challenging drug targets including RNA. In this poster we introduce the method of SMM, the technical process of immobilising SMs onto an SMM chip by non-contact printing, the screening workflow, and data analysis. We demonstrate the technique with a SM screen against distinct protein and RNA targets and show differential SMM binding profiles for each, alongside published SM hits including the SM binders for miR-21-hp RNA and BS-PreQ1 riboswitch RNA. The data presented in this example was collected by one scientist in full within 3 days of hands-on laboratory time, illustrating the accessibility and efficiency of the SMM workflow. INTRODUCTION In SMM, compound libraries are miniaturised and immobilised onto chips for primary drug screening. With specialist array instrumentation hundreds of SMM chips can be manufactured efficiently, and then stored until needed for testing. SMM is compatible with various target types including proteins, structural RNA, DNA and cell lysates. The SMM workflow follows a biochemical binding assay protocol, as outlined in Figure 1. First, the glass chip surface is functionalised, typically with an isocyanate coating, to be highly reactive and allow the immobilisation of a diverse range of SMs from a selected compound library. No library preparation is necessary, as SMM uses unmodified compounds directly from standard library formats and concentrations, typically 10 mM in 100% DMSO. Through microarray printing, each SM in the library is covalently coupled to the chip surface as an individual spot in a spatially resolved microscopic 2D grid. The SMM chip is then incubated with a target of interest, thereby screening for binding against the whole library in parallel. Binding interactions are identified through detection of target molecule fluorescence signal at a discrete spatial position on the microscopic grid. With advances in imaging technologies, data processing is streamlined, meaning hit identification and quantitative binding data can be generated rapidly. SMM binding assay: For recombinant protein binding SMM chips were processed with His-tag protein, following the adapted protocol from Bradner 2006. Protein binding SMM chips were blocked with BSA and incubated with Recombinant Human FKBP12 (Abcam ), DengV-his (SinoBiological ), WDR5-his protein and detected with Alexa Fluor® 647 AffiniPure Donkey Anti-Human IgG (H+L) and Fisher Alexa Fluor® 633 Goat anti-mouse antibody, washing in PBST. Cell lysate detection assay used cell lysate from H522 containing FKBP12-his. The SMM chips were scanned at PMT 35 using the 635 nm channel of an Innopsys Inc. 710 AL scanner. All data was extracted using Mapix® v8.5.0 from Innopsys Inc . For RNA binding assay the protocol was adapted from Connelly, 2017 & 2019 using folded cy5 labelled RNA oligos (IDT ) and scanning at PMT 35 using the 635 nm channel of an Innopsys Inc , 710 AL scanner. Fluorescent intensity values from each SMM screen were extracted using Mapix 8.5.0 (Innopsys Inc ), and SNR values used for data plotting and analysis. REFERENCES 1. Bradner, J. E., McPherson, O. M., Mazitschek, R., Barnes-Seeman, D., Shen, J. P., Dhaliwal, J., Stevenson, K. E., Duffner, J. L., Park, S. B., Neuberg, D. S., Nghiem, P., Schreiber, S. L., & Koehler, A. N. (2006). A Robust Small-Molecule Microarray Platform for Screening Cell Lysates. Chemistry & Biology, 13(5), 493–504. 2. Connelly CM, Boer RE, Moon MH, Gareiss P, Schneekloth J. Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays. ACS Chem Biol. 2017 Feb 17;12(2):435–43. 3. Connelly, C. M., Numata, T., Boer, R. E., Moon, M. H., Sinniah, R. S., Barchi, J. J., Ferré-D’Amaré, A. R., & Schneekloth, J. S. (2019). Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure. Nature Communications 2019 10:1, 10(1), 1–12. 4. Noblin DJ, Page CM, Tae HS, Gareiss PC, Schneekloth JS, Crews CM. A HaloTag-based small molecule microarray screening methodology with increased sensitivity and multiplex capabilities. ACS Chem Biol. 2012 Dec 21;7(12):2055–63. 5. Zhu C, Zhu X, Landry JP, Cui Z, Li Q, Dang Y, et al. Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry. Sensors. 2016 Mar 16;16(3). SM PRINTING & IMMOBILISATION SMM is highly flexible to a variety of SM input library scales, as SMM library chips are batched produced by Arrayjet Mercury printing technology and can be stored ready-to-use for rapid screening of SM binding interactions in a matter of hours. Arrayjet technology uses less than 0.5 µL of each SM to batch produce > 250 ready to use SMM chips for screening. The typical input into a SMM production is a library of SM at stock 10 mM concentrations in 100% DMSO, and both pre-existing customer libraries or Arrayjet internal SM libraries can be accommodated. Surface coupling via isocyanate groups is attractive due to high reactivity for binding the broad range of nucleophilic chemical groups commonly found in small molecule libraries, meaning large pre-existing libraries can be quickly and efficiently converted into SMM chips (Bradner 2006, Zhu 2016). ARRAYJET SMM-10: AN ACCESSIBLE PILOT SMM SCREEN Until now SMM has been the reserve of a minority of groups with specialised expertise and equipment. Arrayjet has democratised access to SMM by taking care of all the up-stream work and placing an easy-to-use screening kit with reagents and a protocol directly in the hands of discovery researchers. Working with medicinal chemist specialists, Arrayjet has used a machine learning model to select an RNA focused library. The Arrayjet SMM-10 contains 5000 representative drug like SM selection from our wider RNA focused library, along with a general diversity set of 5000 SM compounds allowing the screen to be used for a diverse range of targets. This 10,000 SM library is pre-printed and immobilised in duplicate on slides (Figure 5) meaning all hits come with two data points for every one-to-one interaction. Each kit contains one screening chip, all reagents and a protocol. SMM-10 is ideal for discovery groups looking for a high-throughput screen with a simplified cost structure. All compounds are commercially available, without IP constraints and with no commercial reach-through on hits. SMM-10 is also ideal for groups working on RNA or protein targets. With the SMM-10 we are making this technique widely accessible as a tool for groups to quickly test the technique before committing to larger screening campaigns. It is available now for pilot screens to be run by you, or for you by Arrayjet. www.arrayjet.com Figure 1: An overview of the SMM process Figure 3: Novel and known SM hits are identified for a variety of target types by SMM. (A) Schematic of diverse target types screened. (B) Representative Cy5 or anti-His-Tag fluorescent images of SMM screen for each target type listed (Innopsys 710AL, & Mapix software). (C) Heatmap comparing scaled SNR values from each SMM chip screen. SNR values represent median fluorescent signal from 6 replicate data points per SM. (D) SM hit SNR values for published SM binders for miR-21-hp RNA, BS-PreQ1 riboswitch RNA, and FKBP12-his target compared to DMSO values. (E) SM compound structures identified in 2,112 SMM screen shown in (D) and associated published references. Figure 2: The SMM chip production process (A) Unmodified small molecule libraries are used as the input to SMM chip printing and can be produced at variety of scales from 100-500,000 compounds. (B) SMM chips are produced using Arrayjet Mercury piezoelectric printing technology. (C) Schematic of the literature standard isocyanate surface chemistry used for the SMM production process, which allows capture of diverse libraries of SM compounds through attachment via nucleophilic chemical groups (Zhu 2016). Figure 4: Workflow steps with timings for the SMM process PROCESS STEP DURATION (hrs) DETAIL SET-UP 2 Plate and instrument preparation PRINTING 12 (overnight) Arrayjet Mercury-100 microarrayer (15 chips x 12,672 SM) BLOCKING 2 Washing & blocking surface RNA ASSAY 2 Binding assay, imaging PROTEIN ASSAY 5 Binding assay, detection, imaging LYSATE ASSAY 5 Binding assay, detection, imaging ANALYSIS 2 Image analysis, hits identification METHOD: A SMM SCREEN AGAINST 5 TARGETS SMM chip production: 2112 unique small molecule samples in 100% DMSO and control compounds (fluorescent dyes, control compounds and biotin control molecules) were printed (200pl) in 6 technical repeats per chip on a Mercury 100 by Arrayjet Ltd. SM were printed on an isocyanate functionalised glass slide as described by Bradner 2006, Zhu 2016. Each SMM chip contained 12672 total spots. All chips were blocked for immobilisation following an adapted protocol from Connelly et al., 2019. A biotin-streptavidin detection assay was performed to QC the SMM chip printing and SM immobilisation process, using Stratech IgG Fraction Monoclonal Mouse Anti-Biotin and Fisher Alexa Fluor® 633 Goat anti-mouse antibody for detection, imaging performed using Innopsys Inc , 710 AL scanner. Figure 5: Image of a SMM library-on-a-chip SMM chip printed with 10,000 fluorescent small molecules in duplicate, representative of the Arrayjet SMM-10. Each chip is 75 x 25 mm.