Combined Use of Extract Libraries and HPLC-Based Activity Profiling for Lead Discovery: Potential, Challenges, and Practical Considerations

Abstract

Introduction !The unique contribution of natural products to drug discovery is undisputed. Approximately 35 % of the new chemical entities approved as drugs over the past 30 years are directly derived from natural products [1]. However, despite this impressive track record, we have witnessed over the past two decades in the pharmaceutical industry a steady decline of interest for natural products. Various factors have contributed to this apparently paradoxical situation. Emerging technologies may have appeared at one time more promising and "easy" than natural products, which are often perceived as "old-fashioned" at the top management level. However, there have also been more objective reasons for this decline. With the advent of high throughput screening (HTS) in the 1990s, natural product research struggled to find its place in the new drug discovery environment. The classical approach relying on several iterative steps of bioactivity-guided fractionation could not keep pace with the fast turnaround and the tight deadline of HTS-based screening programs [2]. At the same time, the technological advances in HTS, in particular with respect to automation and data management, and significant developments in chromatography and spectroscopy have opened entirely new possibilities for the investigation of natural product extracts. In a time where most major pharmaceutical companies have discontinued their natural product screening activities, these new opportunities have been recognized by biotech companies, and by an increasing number of academic research groups. Switching to a database format for extract handling enables efficient library management and smooth data transfer to screening facilities. The use of new strategies combining, in a more or less seamless manner, HPLC microfractionation with spectroscopic and bioactivity data enables prioritization of hits and identification of bioactive constituents at an early stage [2]. In this context, we set up a tailored technology platform for library-based lead discovery. Its core features are an extract library in 96-well format combined with a customized database, automated liquid handling, HPLC-based tracking of bioactivity, on-line (DAD and ESI-and APCI-MS, including HR-MS) and off-line (microprobe NMR) spectroscopy for structure elucidation. We describe here the different components of this platform and discuss the potential of our workflow Abstract ! A 96-well format plant extract library and a tailored technology platform have been set up for the discovery of new natural product lead compounds. The considerable advantages of the library approach are discussed. Key considerations such as sample generation, logistics, and data management are addressed. The potential of a HPLC-based profiling approach combining offline bioassays with in-line and off-line spectroscopy (including HR-ESIMS and microprobe NMR) for tracking bioactivity is demonstrated with a selection of examples encompassing different types of bioassay formats. The information generated by this approac...

hERG Channel Inhibitory Daphnane Diterpenoid Orthoesters and Polycephalones A and B with Unprecedented Skeletons from Gnidia polycephala

“…Hence, the structure of 11 was assigned as 2β-hydroxy-1βH,7αH,10αH-guaia-4,11-(12)-dien-3-one, the C-10 epimer of the reported stereoisomer. 27 Compound 12 had a molecular formula of C 15 H J coupling and NOESY associations confirmed the same relative configurations at carbons C-1, C-2, C-7, and C-10 as in 11 ( Figure 4B). The α-orientation of the C-6 hydroxy function was inferred from a 3 J H6, H7 value of 9.3 Hz, which implied an anti-diaxial orientation of these protons.…”

“…15 The chromatogram (254 nm) of a semipreparative HPLC separation and the corresponding activity profile of the time-based microfractionation are shown in Figure 1. By means of on-line (UV) and off-line (MS and microprobe NMR) data analyses bioactivity was correlated with peaks in the chromatogram.…”

“…The ROESY spectra of 3 and 4 (Supporting Information) and their positive optical rotation confirmed their configurations as reported. 26 Compound 11 showed a molecular formula of C 15 ]. These spectroscopic data were readily assigned to a guaiane-type sesquiterpenoid with the same planar structure as 2β-hydroxy-1βH,7αH,10βH-guaia-4,11-(12)-dien-3-one reported from the roots of Ligularia narynensis (Asteraceae).…”

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I

“…One library contained 70 pure natural compounds as 10 mM solutions in DMSO, and a second library consisted of 2128 extracts from plants and fungi archived as 10 mg/ml solutions in DMSO (32). Harmine hydrochloride was purchased from TCI Europe (H0002).…”

“…To identify the active principles in these extracts, we used an approach referred to as HPLC-based activity profiling. It combines the separation of complex mixtures with spectroscopic data recorded online and with biological information obtained in parallel from time-based microfractionation and a subsequent bioassay (32). In addition, offline microprobe NMR analysis was used to fully establish the structure of active compounds.…”