Virtual screening of potential anticancer drugs based on microbial products.

The development of microbial products for cancer treatment has been in the spotlight in recent years. In order to accelerate the lengthy and expensive drug development process, in silico screening tools are systematically employed, especially during the initial discovery phase. Moreover, considering the steadily increasing number of molecules approved by authorities for commercial use, there is a demand for faster methods to repurpose such drugs. Here we present a review on virtual screening web tools, such as publicly available databases of molecular targets and libraries of ligands, with the aim to facilitate the discovery of potential anticancer drugs based on microbial products. We provide an entry-level step-by-step description of the workflow for virtual screening of microbial metabolites with known protein targets, as well as two practical examples using freely available web tools. The first case presents a virtual screening study of drugs developed from microbial products using Caver Web, a web tool that performs docking along a tunnel. The second case comprises a comparative analysis between a wild type isocitrate dehydrogenase 1 and a mutant that results in cancer, using the recently developed web tool PredictSNPOnco. In summary, this review provides the basic and essential background information necessary for virtual screening experiments, which may accelerate the discovery of novel anticancer drugs.

Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase.

Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme's specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.

Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method.

Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3Cpro) towards improved proteolytic activity on the substrate LEVLFQ↓GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3Cpro library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

Affinity Maturation of a Cyclic Peptide Handle for Therapeutic Antibodies Using Deep Mutational Scanning.

Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging, and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning. Deep sequencing was used to construct a fitness landscape of this protein-peptide interaction, and four mutations were identified that together improved the affinity for cetuximab 10-fold to 15 nm Importantly, the increased affinity translated into enhanced cetuximab-mediated recruitment to EGF receptor-overexpressing cancer cells. Although in silico Rosetta simulations correctly identified positions that were tolerant to mutation, modeling did not accurately predict the affinity-enhancing mutations. The experimental approach reported here should be generally applicable and could be used to develop meditope peptides with low nanomolar affinity for other therapeutic antibodies.

Biomarkers in Medicines Development-From Discovery to Regulatory Qualification and Beyond.

Biomarkers are important tools in medicines development and clinical practice. Besides their use in clinical trials, such as for enrichment of patients, monitoring safety or response to treatment, biomarkers are a cornerstone of precision medicine. The European Medicines Agency (EMA) emphasised the importance of the discovery, qualification, and use of biomarkers in their Regulatory Science Strategy to 2025, which included the recommendation to enhance early engagement with biomarker developers to facilitate regulatory qualification. This study explores the journey of biomarkers through the EU regulatory system and beyond, based on a review of interactions between developers and the EMA from 2008 to 2020, as well as the use of qualified biomarkers in clinical trials. Of applicants that used early interaction platforms such as the Innovation Task Force, less than half engaged in fee-related follow-up procedures. Results showed that, as compared to companies, consortia were more likely to opt for the Qualification of Novel Methodologies procedure and engage in follow-up procedures. Our results highlight the importance of early engagement with regulators for achieving biomarker qualification, including pre-submission discussions in the context of the qualification procedure. A review of clinical trials showed that all qualified biomarkers are used in practice, although not always according to the endorsed context of use. Overall, this study highlights important aspects of biomarker qualification, including opportunities to improve the seamless support for developers by EMA. The use of qualified biomarkers in clinical trials underlines the importance of regulatory qualification, which will further enable precision medicine for the benefit of patients.

Biomarker Qualification at the European Medicines Agency: A Review of Biomarker Qualification Procedures From 2008 to 2020.

Regulatory qualification of biomarkers facilitates their harmonized use across drug developers, enabling more personalized medicine. This study reviews various aspects of the European Medicines Agency's (EMA's) biomarker qualification procedure, including frequency and outcome, common challenges, and biomarker characteristics. Our findings provide insights into the EMA's biomarker qualification process and will thereby support future applications. All biomarker-related "Qualification of Novel Methodologies for Medicine Development" procedures that started from 2008 to 2020 were included. Procedural data were extracted from relevant documents and analyzed descriptively. In total, 86 biomarker qualification procedures were identified, of which 13 resulted in qualified biomarkers. Whereas initially many biomarker qualification procedures were linked to a single company and specific drug development program, a shift was observed to qualification efforts by consortia. Most biomarkers were proposed (n = 45) and qualified (n = 9) for use in patient selection, stratification, and/or enrichment, followed by efficacy biomarkers (37 proposed, 4 qualified). Overall, many issues were raised during qualification procedures, mostly related to biomarker properties and assay validation (in 79% and 77% of all procedures, respectively). Issues related to the proposed context of use and rationale were least common yet were still raised in 54% of all procedures. While few qualified biomarkers are currently available, procedures focus increasingly on biomarkers for general use instead of those linked to specific drug compounds. The issues raised during qualification procedures illustrate the thorough discussions taking place between applicants and regulators-highlighting aspects that need careful consideration and underlining the importance of an appropriate validation strategy.

Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome.

We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome-a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.

Exploring the therapeutic potential of modern and ancestral phenylalanine/tyrosine ammonia-lyases as supplementary treatment of hereditary tyrosinemia.

Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs. Surprisingly, most putative ancestors could be expressed as functional tetramers in Escherichia coli and thus retained their ability to oligomerize. All ancestral enzymes displayed increased thermostability compared to both modern enzymes, however, the increase in thermostability was accompanied by a loss in catalytic turnover. One reconstructed ancestral enzyme in particular could be interesting for further drug development, as its ratio of specific activities is more favourable towards tyrosine and it is more thermostable than both modern enzymes. Moreover, long-term stability assessment showed that this variant retained substantially more activity after prolonged incubation at 25 °C and 37 °C, as well as an increased resistance to incubation at 60 °C. Both of these factors are indicative of an extended shelf-life of biopharmaceuticals. We believe that ancestral sequence reconstruction has potential for enhancing the properties of enzyme therapeutics, especially with respect to stability. This work further illustrates that resurrection of putative ancestral oligomeric proteins is feasible and provides insight into the extent of conservation of a functional oligomerization surface area from ancestor to modern enzyme.

Ancestral diterpene cyclases show increased thermostability and substrate acceptance.

Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7-13 °C. Ancestral spiroviolene synthases catalyze cyclization of the natural C20 -substrate geranylgeranyl diphosphate (GGPP) and also accept C15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate-rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., kcat /KM for GGPP over kcat /KM for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.