Drugging the ‘undruggable’: new technology for drug candidate identification 11th August 2021
Oxford, UK based Orbit Discovery has developed a novel bead-based and microfluidic technology platform that screens peptide and small-molecule libraries for target binding and subsequent activity in cell-based functional assays, thus enabling the company to identify new drug candidates active against previously ‘undruggable’ targets.
Functional screening service company Orbit Discovery, which has recently raised $7.6 million in funding from Oxford Sciences Innovation and other institutional and individual investors, is now expanding its activities as a provider of peptide discovery services to the biotechnology and pharmaceutical industry: the company has developed a novel bead-based and microfluidic technology platform that enables the screening of peptide and other compound libraries for target binding and subsequent activity in cell-based functional assays, thus identifying peptide or other candidate compounds active against both soluble proteins and membrane-bound targets such as GPCRs.
Affinity and functionality screens
According to the company, until now, researchers have had difficulty in combining high throughput affinity screens with functional screens utilizing peptide libraries of very high chemical diversity. The use of a bead-based peptide display engine along with microfluidic technologies allows Orbit to interrogate the activity of each library member against single cells which enables the identification of functional molecules including those that may have agonist or antagonist properties on disease-relevant targets. These functional leads provide the starting point for the development of peptide, small-molecule or other therapeutics or bioconjugates, providing precisely targeted cargo delivery mechanisms.
Orbit says it aims to become the leader in functional screening by using a combination of its screening know-how and miniaturization to improve throughput and identify rare hits with the desired biological function, combining and translating its technologies for the development of breakthrough human therapeutics for its clients, which include both large pharmaceutical companies and small to medium-sized biotechs.
Target purification: the key to success
The purification of a suitable target for any screening process is the key to success and Orbit offers the expertise to design, construct and validate a target and accessory proteins to present them in a number of possible forms: as native protein; in artificial membranes; and in cell models. Orbit says that whichever of the techniques is adopted ensures the creation of functional target proteins that can be used to identify peptides that act as the starting point for further drug development. The company has also developed specific expertise in purifying multi-membrane spanning proteins (GPCRs, in particular) that maintain a functional state after purification and develops assays specifically for the developed target to facilitate identification of therapeutic peptides during the screening processes.
In addition to carrying out this target purification, the design and construction of screening libraries can be performed using the company’s internal knowledge on structure, diversity and modelling from known motifs.
The screening process
In a typical drug discovery project, target material generated by Orbit or supplied by the client is screened using novel FACS based methodologies, and target material can come in many formats including soluble protein; in situ cell-bound protein; or within an artificial membrane in a form activated by associated accessory proteins.
Proteins are screened against libraries of tens of billions of peptides in order to identify panels of specific target binders that can then be ranked for their intrinsic affinity to the target and form the scaffold for further optimization and development as therapeutic candidates. If a functional screen is required, the binders can be further analyzed for using cell-based assays that employ either plate-based screening or droplet-based microfluidics.
With plate-based techniques, libraries of up to 50,000 peptides derived from an affinity screen can be screened against a target presented in one or more functional cell models and generic activation of the target function is demonstrated directly in the screen, enabling hits to be selected and sorted.
When assessing rarer events, or when a large number of leads is required, the company’s microfluidic and droplet-based techniques can be used to screen libraries of up to one million peptides, providing an ideal way to search for agonistic and antagonistic functional peptides and allowing the effect of the peptides on real life read-outs to be built into a screening cascade.
Microbead technology: the details
The new technology developed by Orbit presents DNA and peptides on the surface of a microbead, allowing the creation of unbiased libraries and with a tunable multivalent display allows access to very broad chemical diversity. This can handle unnatural amino acids as well as complex peptides and can display a peptide via complex scaffolds thereby making whole-cell screening and functional hits possible.
The bead based system carries DNA constructs and peptides encoded by those constructs with a single DNA/peptide species per bead linking genotype to phenotype. This single-codon DNA methodology avoids problems of codon bias with quick and inexpensive synthesis. Bead sizes and the range of bead types used are flexible enough to allow for focused platform application development, the peptide display being highly configurable in number from 10s to 10,000s per bead. The peptide conformation can be tailored from linear to cyclic to complex macrocycles and any size of peptide can be displayed, although typically these are in the range of 5 to 30 AAs. Unnatural amino acids can be incorporated and post translational modifications made.
A peptide display engine using in vitro transcription-translation (IVTT) display of peptides on beads delivers a broad range of capabilities and is able to address soluble targets and targets in situ on and in cells thus allowing for significantly faster time to discovery of relevant peptide leads based on affinity screens and/or functional screens. The ability to display high peptide copy number results in the novel application of screening complex peptide libraries by use of cell-based functional assays, which brings the discovery of functional peptides to the primary screen rather than after months (or years) of deconvolution of simple binders.
Tackling difficult targets
The Orbit platform is target agnostic but highly capable of tackling difficult targets. Orbit has expertise with soluble proteins and cell surface receptors, notably GPCRs and ion channels, enabling the screening of peptides that can address currently difficult or ‘undruggable’ protein-protein interactions.
Screening workflows are aligned with target type and desired characteristics of hit molecules and Orbit has developed a range of novel selection methodologies ranging from simple biopanning to more complex affinity screens, FACS based selection and sorting for functional screening and single-cell analysis, which is important for throughput of primary screens and in later screens where primary cells are required and cell numbers may become limiting. The high throughput screening technology improves the probability of a hit on each screening round.
The use of microfluidics allows for the miniaturization of library production and cellular screening to enable complex screens to be performed at relevant scale. The microfluidic capability allows for fine control over library generation and for screening and manipulation of single cells in droplets.
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