Intracellular targeting with engineered proteins

If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action.

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Toxin Neutralization Using Alternative Binding Proteins

Toxins ◽

10.3390/toxins11010053 ◽

2019 ◽

Vol 11 (1) ◽

pp. 53 ◽

Cited By ~ 10

Author(s):

Timothy Jenkins ◽

Thomas Fryer ◽

Rasmus Dehli ◽

Jonas Jürgensen ◽

Albert Fuglsang-Madsen ◽

...

Keyword(s):

Binding Protein ◽

Low Cost ◽

Protein Scaffolds ◽

Comprehensive Overview ◽

Alternative Protein ◽

Human Proteins ◽

Cost Of Production ◽

High Level ◽

Antibody Derivatives ◽

Derivatives Of

Animal toxins present a major threat to human health worldwide, predominantly through snakebite envenomings, which are responsible for over 100,000 deaths each year. To date, the only available treatment against snakebite envenoming is plasma-derived antivenom. However, despite being key to limiting morbidity and mortality among snakebite victims, current antivenoms suffer from several drawbacks, such as immunogenicity and high cost of production. Consequently, avenues for improving envenoming therapy, such as the discovery of toxin-sequestering monoclonal antibodies against medically important target toxins through phage display selection, are being explored. However, alternative binding protein scaffolds that exhibit certain advantages compared to the well-known immunoglobulin G scaffold, including high stability under harsh conditions and low cost of production, may pose as possible low-cost alternatives to antibody-based therapeutics. There is now a plethora of alternative binding protein scaffolds, ranging from antibody derivatives (e.g., nanobodies), through rationally designed derivatives of other human proteins (e.g., DARPins), to derivatives of non-human proteins (e.g., affibodies), all exhibiting different biochemical and pharmacokinetic profiles. Undeniably, the high level of engineerability and potentially low cost of production, associated with many alternative protein scaffolds, present an exciting possibility for the future of snakebite therapeutics and merit thorough investigation. In this review, a comprehensive overview of the different types of binding protein scaffolds is provided together with a discussion on their relevance as potential modalities for use as next-generation antivenoms.

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Alternative Protein Scaffolds for Molecular Imaging and Therapy

Engineering in Translational Medicine ◽

10.1007/978-1-4471-4372-7_13 ◽

2013 ◽

pp. 343-364

Author(s):

Benjamin J. Hackel

Keyword(s):

Molecular Imaging ◽

Protein Scaffolds ◽

Alternative Protein

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Alternative Protein Scaffolds as Novel Biotherapeutics

Biobetters - AAPS Advances in the Pharmaceutical Sciences Series ◽

10.1007/978-1-4939-2543-8_13 ◽

2015 ◽

pp. 221-268 ◽

Cited By ~ 5

Author(s):

Michaela Gebauer ◽

Arne Skerra

Keyword(s):

Protein Scaffolds ◽

Alternative Protein

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Beyond antibodies ? lessons from bacterial ?immunity?

Microbiology Australia ◽

10.1071/ma06080 ◽

2006 ◽

Vol 27 (2) ◽

pp. 80

Author(s):

Stewart D Nuttall ◽

Suzy M Juraja ◽

Jennifer A Carmichael

Keyword(s):

Binding Proteins ◽

Specific Binding ◽

Immune Surveillance ◽

Specific Protein ◽

Protein Scaffolds ◽

Immune Systems ◽

Alternative Protein ◽

Human Proteins ◽

Specific Binding Molecules ◽

Highly Evolved

Isolation and production of highly specific protein-based binding molecules are crucial to the ever expanding diagnostics, therapeutics and protein array fields. Traditionally, such reagents have been sourced from vertebrate immune systems, where antibodies have evolved over millennia into highly effective molecules of immune surveillance capable of targeting a huge range of targets in response to infection and disease. Now, a growing number of alternative protein scaffolds are being investigated as specific binding molecules incorporating a diverse and powerful range of binding and recognition interfaces. These are being sourced from human proteins, from alternative immune molecules present in evolutionarily old vertebrates, and from highly evolved binding proteins in prokaryotic systems.

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Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

Biochemical Journal ◽

10.1042/bcj20190812 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3835-3847 ◽

Cited By ~ 1

Author(s):

Aliyath Susmitha ◽

Kesavan Madhavan Nampoothiri ◽

Harsha Bajaj

Keyword(s):

Protein Engineering ◽

Cell Attachment ◽

Functional Characterization ◽

Protein Structures ◽

Structural Similarity ◽

Surface Proteins ◽

Sortase A ◽

Peptide Cleavage ◽

New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

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