Proteins come in all shapes and sizes, ranging from thousands of amino acids in length to less than 20. However, smaller size does not correlate with reduced importance. Miniproteins, which are commonly defined as being less than 100 amino acids long, are receiving increased attention for their potential roles as pharmaceuticals. A recent paper by David Baker’s group put miniproteins into the spotlight, as the study authors were able to design miniproteins that bind the SARS-CoV-2 spike protein with as strong affinity as an antibody would – but in a tiny fraction of the size (Cao et al., 2020). These miniproteins are much cheaper to manufacture than antibodies (as they can be expressed in bacteria) and can be highly stable (with melting temperatures of >90º possible, meaning they can easily be stored at room temperature). The most promising miniprotein developed by the Baker group (LCB1) is currently undergoing testing to be used as a prophylactic nasal spray that provides protection against SARS-CoV-2 infection. These promising results – and the speed in which progress was made – brings the vast potential of miniproteins in healthcare to the fore.

Miniproteins in nature

Miniproteins, also known as microproteins or micropeptides, are not just a creative re-imagination of proteins – they exist in nature and play important roles across organisms. While they were traditionally overlooked, as researchers typically excluded proteins with less than 100 amino acids from analyses (Leslie, 2019), more attention has been paid to these ‘pint-sized’ proteins in recent years. The exact numbers of miniproteins expressed in different organisms remains unclear, but studies suggest it could be on the order of hundreds or even thousands (Aspden et al., 2014; Bazzini et al., 2014; Sberro et al., 2019). The miniproteins that have been identified to date may only represent a fraction of expressed miniproteins, yet even these are highly diverse in function. One common theme in their mode of action is that, due to their small size, miniproteins often have regulatory functions that are mediated through interactions with larger proteins (Leslie, 2019).

Various miniproteins that have been identified act by regulating membrane protein channels and transporters. For example, miniproteins in the venoms of various species bind to and block ion channels, thereby impairing the nerve function of the victim (Leslie, 2019). E. coli resistance to antibiotic drugs is aided by a miniprotein: AcrZ stimulates an efflux pump that exports drugs out of the cell (Figure 1) (Hobbs et al., 2012). Miniproteins also act at a more complex level in tissue development and function. The normal development of legs in Drosophila requires a very short protein, Tal, just 11 amino acids (Pueyo & Couso, 2008). In mouse muscles, a trio of miniproteins, each less than 55 amino acids, regulate muscle contraction – the deletion of one of these, myoregulin, enhanced the mice’s running performance (Anderson et al., 2015).

Synthetic miniproteins

In addition to their important endogenous roles, synthetic miniproteins are receiving more attention as putative therapeutics. Their properties – small size (1-10 kDa) and ability to bind targets with high specificity and affinity – would allow them to fill a gap between small molecule drugs and antibody(-like) biologics. Miniproteins may be able to bind sites (e.g., flat protein-protein interfaces) that have previously been classified as ‘undruggable’ (Crook et al., 2020). They are also easier to manufacture and administer than antibody therapeutics.

There are numerous miniproteins in development for healthcare applications, although few have reached the final stages or approval yet. These miniproteins have varied functions: in addition to agonists, antagonists, and protein-protein interaction disruptors, miniproteins are being used as ‘ferries’ (delivering e.g., small molecule drugs or molecules that can help with diagnosis to a site of interest, such as a tumour) and ‘joiners’ (bringing targets together in space e.g., when fused to an antibody) (Crook et al., 2020).

Natural miniproteins have been the inspiration for multiple pharmaceuticals. One exciting example is a miniprotein, naturally found in the deathstalker scorpion (Leiurus quinquestriatus), that has been repurposed for tumor identification and removal. This miniprotein, chlorotoxin, surprisingly binds isoforms of matrix metalloproteinase-2 (MMP-2) that are specifically found in glioma brain tumours (Deshane et al., 2003). Researchers at The Fred Hutchinson Cancer Research Center led by Dr. Jim Olson fused a modified chlorotoxin with a fluorescent dye. This fusion, which is aptly named “Tumor Paint”, is in clinical trials to assess its ability to help visualize tumours and their surgical removal (Leary et al., 2020).

There are also entirely novel miniproteins, which are not strictly based on anything that exists in nature. The SARS-CoV-2–binding miniproteins developed in the Baker group at the Institute for Protein Design, University of Washington mentioned at the start of this post fall in this category (Cao et al., 2020). The authors of the study tried two methods for generating high affinity binders against the SARS-CoV-2 spike protein: developing a miniprotein (1) which includes the helix of the ACE2 receptor which is known to interact with the spike protein and (2) from scratch, with no predetermined scaffold. For both methods, they generated initial constructs computationally then validated these and improved binding experimentally. Interestingly, Cao et al. achieved greater success via the second method; this may be because a greater diversity of structures (including ones that improve binding affinity) can be sampled without the constraint of method 1 (Cao et al., 2020). In the end, they were able to generate two miniproteins that bind the SARS-CoV-2 spike protein with sub-nanomolar affinity (Figure 2). They also found that these miniproteins were able to neutralise the SARS-CoV-2 virus with IC50 values between 20-50 picomolar – this is nearly as strong as the most potent monoclonal antibody that has been discovered against SARS-CoV-2 (Cao et al., 2020)!

It is possible to engineer strong and specific binding in miniproteins, a property that is prized in antibody therapeutics. Furthermore, there are numerous possible advantages for pharmaceutical miniproteins, as compared with designed antibodies. For example, miniproteins may be:

• Able to penetrate tissues and cells more easily, due to their small size
• Cheaper to manufacture, as many types of miniproteins can be expressed by bacteria
• Engineered to be very thermostable and therefore can be stored at room temperature (or even be freeze-dried or survive more extreme conditions e.g., boiling)

There have been tremendous advances in protein engineering – and corresponding computational methods – in recent years. Simultaneously, the importance and diversity of small proteins has been explored. Combining ideas from both these categories could be transformative for pharmaceutical development, which is currently hampered by high costs and low success rates. Miniprotein design will be an exciting space to watch!

References

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