Identification and engineering of potent cyclic peptides with selective or promiscuous binding through biochemical profiling and bioinformatic data analysis.

As our understanding of biological systems grows, so does the need to selectively target individual or multiple members of specific protein families in order to probe their function. Many targets of current biological and pharmaceutical interest are part of a large family of closely related proteins and achieving ligand selectivity often remains either an elusive or time-consuming endeavour. Cyclic peptides (CPs) occupy a key niche in ligand space, able to achieve high affinity and selectivity while retaining synthetic accessibility. De novo cyclic peptide ligands can be rapidly generated against a given target using mRNA display. In this study we harness mRNA display technology and the wealth of next generation sequencing (NGS) data generated to explore both experimental approaches and bioinformatic, statistical data analysis of peptide enrichment in cross-screen selections to rapidly generate high affinity CPs with differing intra-family protein selectivity profiles against fibroblast growth factor receptor (FGF-R) family proteins. Using these methods, CPs with distinct selectivity profiles can be generated which can serve as valuable tool compounds to decipher biological questions.

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans.

Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR-Cas9-based 'gene drive array' platform to facilitate efficient genetic analysis in C. albicans. In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens.

Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines.

Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII productivities was selected for RNA sequencing analysis. The analysis showed distinct differences in F8 RNA composition between the clones. The exogenous F8-dhfr transcript was found to make up the most abundant transcript in the present clones. No correlation was seen between F8 mRNA levels and the measured FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce the mature mRNA composition of the transgene and identify significant truncations that would probably otherwise have remained undetected.

Adaptive evolution of drug targets in producer and non-producer organisms.

MPA (mycophenolic acid) is an immunosuppressive drug produced by several fungi in Penicillium subgenus Penicillium. This toxic metabolite is an inhibitor of IMPDH (IMP dehydrogenase). The MPA-biosynthetic cluster of Penicillium brevicompactum contains a gene encoding a B-type IMPDH, IMPDH-B, which confers MPA resistance. Surprisingly, all members of the subgenus Penicillium contain genes encoding IMPDHs of both the A and B types, regardless of their ability to produce MPA. Duplication of the IMPDH gene occurred before and independently of the acquisition of the MPAbiosynthetic cluster. Both P. brevicompactum IMPDHs are MPA-resistant, whereas the IMPDHs from a non-producer are MPA-sensitive. Resistance comes with a catalytic cost: whereas P. brevicompactum IMPDH-B is >1000-fold more resistant to MPA than a typical eukaryotic IMPDH, its kcat/Km value is 0.5% of 'normal'. Curiously, IMPDH-B of Penicillium chrysogenum, which does not produce MPA, is also a very poor enzyme. The MPA-binding site is completely conserved among sensitive and resistant IMPDHs. Mutational analysis shows that the C-terminal segment is a major structural determinant of resistance. These observations suggest that the duplication of the IMPDH gene in the subgenus Penicillium was permissive for MPA production and that MPA production created a selective pressure on IMPDH evolution. Perhaps MPA production rescued IMPDH-B from deleterious genetic drift.

A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi.

BACKGROUND: Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several Penicillium species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from Penicillium brevicompactum revealed an extra copy of the IMPDH-encoding gene (mpaF) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by mpaF. RESULTS: In accordance with our hypothesis, heterologous expression of mpaF dramatically increased MPA resistance in a model fungus, Aspergillus nidulans, which does not produce MPA. The growth of an A. nidulans strain expressing mpaF was only marginally affected by MPA at concentrations as high as 200 µg/ml. To further substantiate the role of mpaF in MPA resistance, we searched for mpaF orthologs in six MPA producer/non-producer strains from Penicillium subgenus Penicillium. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting mpaF homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class. CONCLUSIONS: We identified a novel variant of the IMPDH-encoding gene in six different strains from Penicillium subgenus Penicillium. The novel IMPDH variant from MPA producer P. brevicompactum was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.