High throughput screening (HTS) has been the go-to technique for early-stage drug discovery for many years. Researchers use it for the rapid identification of large numbers of active compounds, which are then assessed by their ability to influence a particular biological pathway and to emerge as a new drug.
The Griffith Institute for Drug Discovery (GRIDD), Griffith University, Brisbane, is an example of one laboratory using HTS robotics for automated screening, to increase the overall speed of drug discovery. Complementary to HTS is fragment based drug discovery (FBDD). FBDD has emerged as a powerful tool for discovering drug leads. This approach must first identify very small molecules (fragments) that bind to proteins. GRIDD focuses on combining mass spectrometry (MS) with FBDD, to observe fragment-protein interactions to identify these superior starting points for drug discovery.
Introducing mass spectrometry to drug discovery
The team at GRIDD has significantly improved its fragment screening capabilities, by investing in Magnetic Resonance Mass Spectrometry (MRMS), previously known as Fourier Transform Mass Spectrometry (FTMS). With a recent infrastructure upgrade the upper size limit of proteins that can be studied in their native state has increased from 50 to 150 kilodaltons (kDa), which has made many more proteins of interest accessible for downstream drug development.
The accurate identification of the binding and molecular mass for fragments is a very important starting point in drug discovery. The extreme resolution afforded by MRMS provides an extra layer of confidence in fragment screening as protein-fragment interactions are very weak interactions.
Whereas other fragment screening techniques suffer from producing high false positives as they are having to deal with the fragments at high concentrations, MRMS produces minimal false positives. When fragments are at higher concentrations, they can aggregate and behave irregularly. When using MRMS, the fragment is at a similar concentration as the protein, so false hits are less likely to occur, which is highly advantageous. The MRMS method can quickly rule out ineffective compounds to avoid wasting valuable time on compounds that will not advance along the discovery and development pipeline.
Progressing through the pipeline with FBDD
The investment in small molecules to progress them to phase I, II and III clinical trials, before failing late in the process is referred to as attrition, and is extremely costly. Drug discovery researchers are increasingly looking towards FBDD to lower attrition rates and improve the rate of acceptance of drug leads and progression through the drug development process or ‘pipeline’.
Molecular size is the key parameter that differentiates a fragment and traditional HTS compound. A fragment usually has a molecular weight of under 200 daltons (Da), whereas a HTS compound is between 400-500 Da. Fragments have a weaker binding affinity to proteins than HTS compounds, but if an interaction does occur, it indicates a perfect fit. Therefore, compounds identified by fragment screening are likely to progress further in the drug development pipeline towards Food and Drug Administration (FDA) approval and lower attrition rates.
Bringing screening technologies together
There have been three approved drugs from the fragment screening technique, Vemurafenib (also known as Zelboraf), Venetoclax and Kisqali® (LEE011, ribociclib), with many more in phase I, II and III clinical trials [1]. FBDD is, however, dependent on analytical method development, such as MS, to identify the weak binding fragments. Researchers at GRIDD are therefore optimizing their workflows and combining techniques together with collaborators in order to accelerate drug discovery. Surface Plasmon Resonance (SPR), X‑ray Crystallography, Nuclear Magnetic Resonance (NMR) and Isothermal Titration Calorimetry (ITC) are additional tools for FBDD, and institutes such as GRIDD are finding the most successful combinations of these methods.
The group combined native state MS with two fragment screening methods, SPR and X-ray crystallography, in a campaign against human carbonic anhydrase II (CA II) [2], an enzyme which catalyzes the hydration of carbon dioxide. Defects in this enzyme are associated with diseases such as osteopetrosis (or “stone bone”) and renal tubular acidosis [3]. Native state MS offers a rapid, sensitive, high throughput, and label-free method to directly investigate protein−ligand interactions, but there have been few studies using this approach as a screening method to identify relevant protein−fragment interactions in FBDD. The results showed the first fragment screening analysis of electrospray ionization (ESI)-MS and NanoESI-MS using a high resolution Fourier-transform ion cyclotron resonance (FTICR) instrument (Bruker solariX XR 12.0T MRMS) in parallel with SPR (Table 1 and Figure 1).
Table 1: Correlation of screening results for fragment hit chemotypes with surface plasmon resonance (SPR), electrospray ionization-mass spectrometry (ESI-MS), NanoESI-MS, and X-ray crystallography (green tick = hit, red cross = not a hit, n/a fragment not tested). aDose-response experiment performed at 25 °C with a 5-point fragment concentration series range. bnano MS hit with ratio of unbound CA II:fragment bound CA II peak intensities in brackets.
The high speed and minimal sample concentration required for MS is this reason for its use as a pre-filtering tool at the front end of the fragment screening cascade of methods at GRIDD.
Quantitative native MS
The group at GRIDD has recently identified a new zinc binder fragment, which is a potent inhibitor of CA II, in collaboration with the Commonwealth Scientific and Industrial Research Organization (CSIRO) [4]. Using SPR and native ESI-MS, the group identified compound 10, which has an affinity and ligand efficiency approaching that of sulfonamides, a well-known class of zinc binder for CA II. By determining the crystal structure of compound 10 bound to CA II (Figure 2), the group could confirm the binding pose of the new fragment to CA II, which included a primary interaction with the zinc and two hydrogen bonds with the protein, therefore explaining the high affinity.
Figure 1: NanoESI mass spectra of hit fragments with carbonic anhydrase II (CA II). The 9+ charge state is shown, with [protein + fragment] 9+ peaks in red. (A) human CA II protein only. (B) 1, primary sulfonamide chemotype. (C) 2, cinnamic acid chemotype. (D) 3, benzoic acid chemotype. (E) 4, phenylacetic acid chemotype. (F) 5, tetrazole chemotype. (G) 6, tetrazole chemotype. (H) 7, 1, 2, 4-triazole chemotype.
Figure 2: Binding interactions of the classic primary sulfonamide chemotype (A) with carbonic anhydrase II (CA II) are emulated by the oxazolidindione chemotype compound 10 (B), discovered by fragment screening using magnetic resonance mass spectrometry (MRMS).
The study used a series of 18 analogues of compound 10 to assess the structure-activity relationship (SAR) using both SPR and MS. The group was able to obtain quantitative MS data by maintaining a constant protein concentration at 14.5 µM, and varying the fragment concentration from 0.5 – 120 µM. Plotting the percentage of protein bound and curve-fitting revealed dissociation constants remarkably similar to those determined using SPR. Nine of the new fragments showed at least some activity, although none were significantly more potent than compound 10. Crystal soaking experiments gave rise to seven new structures, with all fragments binding in a similar manner as compound 10.
FBDD in the future
The growing interest from the pharmaceutical sector in FBDD has led many academic research institutions to focus on industry collaborations. Such industry-academia relationships, in addition to the advances in analytical technology (such as MRMS) have made it possible to discover previously overlooked drug leads.
For more information on the Griffith Institute for Drug Discovery, please visit https://www.griffith.edu.au/institute-drug-discovery or follow on twitter @GRIDD_GU. For more information on the solariX XR, please visit https://www.bruker.com/products/mass-spectrometry-and-separations/ftms/solarix/overview.html.
References
Singh M, Tam B and Akabayov B. (2018) NMR-Fragment Based Virtual Screening: A Brief Overview, Molecules, 23, 233.
Woods L, Dolezal O, Ren B, Ryan J, Peat T, Poulsen S. (2016) Native State Mass Spectrometry, Surface Plasmon Resonance, and X‑ray Crystallography Correlate Strongly as a Fragment Screening Combination, Journal for Medical Chemistry, 59, 5, 2192-2204
Roth DE, Venta PJ, Tashian RE, and Sly WS. (1992) Molecular basis of human carbonic anhydrase II deficiency, Proc Natl Acad Sci USA, 89(5): 1804–1808.
Chrysanthopoulos P, Mujumdar P, Woods L, Dolezal O, Ren B, Peat T and Poulsen S. (2017) Identification of a New Zinc Binding Chemotype by Fragment Screening, Journal for Medical Chemistry, 60, 7333−7349.
About the Author:
Professor Sally-Ann Poulsen, Professor of Chemical Biology at Griffith Institute for Drug Discovery (GRIDD), Griffith University, introduced MRMS (FTMS) use for the study of protein-ligand complexes to Australia, and was one of the first researchers worldwide, and the first in Australia, to utilise native state mass spectrometry to screen fragments by the direct observation of protein-ligand complexes.
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Filed Under: Drug Discovery and Development
