We have understood our aim: to create a sclerostin inhibitor that will both eliminate osteogenesis imperfecta (OI) and allow sclerostin to continue perform its functions to reduce the risk of contracting cardiovascular diseases via inflammation in the arteries. As we've seen with the example of romosozumab, we cannot take a one-size-fits-all solution and just inhibit the entire molecule without question. We must dive deeper.
First, if sclerostin has multiple functions, which parts of the protein is responsible for what function? If we can determine which component is responsible for binding to LRP-5/6 and which for atheroma/aneurysm prevention, then we would only need to target the problematic component to hit two birds with one stone.
Typically, in most proteins, there are four types of structures present:
A polypeptide strand will fold and bend under the right conditions (e.g. surrounding pH, temperature, presence of water and hydrophobic surfaces, etc.) into a protein and gain its tertiary structure. During this process, an arbitrary number of protein loops may form between the N- and C-termini at the ends of the strand as the strand condenses in size. Each protein loop may provide unique binding sites and thus the ability to express different properties to the protein, and each loop may be activated or disabled individually without disrupting the exhibition of other protein loops in the same protein molecule. Note that protein loops are not necessary due to the presence of ω-loops.
Sclerostin has three loops present in its secondary structure[6], illustrated below in Fig E.1:
Fig E.1: 3D NMR structure of sclerostin[6].
Remember the antibody from before? According to Fig. E.2 below, romosozumab binds to human sclerostin via loop 2 and loop 3. Thus, it can be deduced that the loop responsible for causing OI in affected patients and the loop responsible for protection against cardiovascular diseases is loop 2 and loop 3. To assign the properties to the loops, further experiments must be performed.
Fig E.2: A graph showing the affinity of romosozumab to the different loops in sclerostin[6]
To distinguish which loops are responsible for which function of sclerostin, we have created two main types of variants of the sclerostin molecule, one deficient in loop 3 only, and one deficient in both loop 2 and 3, and compared their affinities by administering them to OI mice with Apolipoprotein E deficiency (ApoE-/-).
Fig E.3: Diagram of sclerostin variants and their beneficial suppression effects on the cardiovascular system.
As seen from Fig E.3, the loop 2- and loop 3-deficient sclerostin had lost its suppressive effects on expression of inflammatory cytokines and chemokine in vitro, whereas the loop 3-deficient sclerostin had maintained such functions.
Fig E.4: Demonstration of the cardiovascular effects of sclerostin and its variants.
Not only that, according to Figs E.4, the loop 3-deficient variant sclerostin and the full-length sclerostin both had similar effects in the suppression of activity in inflammatory cytokines and chemokine, and thus the progression of aortic aneurysms and atherosclerosis in mice affected with OI as well. It can thus be concluded that sclerostin's loop 2 is responsible for offering protection against cardiovascular diseases, while loop 3 is responsible for disrupting the Wnt signalling pathway, and thus causing OI.
Great! Now we have our bullseye: loop 3 in a sclerostin molecule. Now all we need is something to inhibit its effects.
Aptamers are a class of functional oligonucleotides which can bind to a wide variety of specific targets, including proteins, peptides, carbohydrates, small molecules, toxins, and even live cells, with high affinities and specificities.
They are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches, and can exist in many different types. A useful distinction between aptamers and normal DNA/RNA strands is that while they can all encode genetic information, aptamers are developed specifically to achieve a high affinity with a specific molecule, receptor or binding site. In a way, they are similar to the many variants of glue (e.g. epoxy, spray adhesives, white craft glue, cyanoacrylate adhesives, etc.) that are well adapted to particular situations.
Their extremely high affinities and other favourable properties, such as ease of administration, lowered costs, reduction of immunosuppressive effects upon and after injection, and many more over other forms of specific treatments (e.g. antibody treatment) make them optimal candidates for this task. For the full list of benefits, please refer to here.
To synthesise the desired aptamer for this project, our team has utilised the SELEX (Sequential Evolution of Ligands by EXponential enrichment) process[7] and modified the aptamer such that the product aptamer will be most suitable for this undertaking.
Fig E.5: General overview of the SELEX process used in this project.
Below is a general overview of the process:
Fig E.6: Affinity data for aptamers in the positive and negative selection rounds in SELEX cycles, and the specificity and affinity data of the Ostamer to sclerostin loop 3.
Comparing the affinities of aptamers in SELEX, the unselected library has the lowest light absorbance, signalling that lots of aptamer sequences did not bind to the positive target well. After the 10th round, the affinity to human sclerostin was mediocre, as shown by the low absorbance rate. After 20 rounds, the affinity to human sclerostin was high.
After extracting the different sequences, we found that one sequence binds to loop 3 of sclerostin while having extremely low affinities for the other loops of sclerostin, and the affinity increased as the concentration of the aptamer increased. This means that this aptamer sequence satisfies the criteria of only binding to loop 3 of sclerostin.
Wonderful! We now have our little aptamer molecule that targets sclerostin's loop 3.
As aptamers are short oligonucleotide sequences, they are cleared quickly in the body via interactions with biomolecules and the kidneys. As a result, the average time of oligonucleotide decay in blood ranges from several minutes to several tens of minutes depending on the oligonucleotide concentration and conformational structure.[8]
Such a short time range is unacceptable for most therapeutic applications, and thus several methods for protecting aptamers against degradation by nucleases have been developed.
For our project, we will use chemical modifications to extend the half-life of the aptamer, similar to the method used by another FDA-approved aptamer-based drug, Macugen[8].
Fig E.7, 8: Chemical modifications added to the ostamer.
[1] Sanger F (1952). "The arrangement of amino acids in proteins". In M.L. Anson; Kenneth Bailey; John T. Edsall (eds.). Advances in Protein Chemistry. 7. pp. 1–67. [2] Linderstrøm-Lang KU (1952). Lane Medical Lectures: Proteins and Enzymes. Stanford University Press. p. 115. [3] Leszczynski, JF; Rose, GD (1986). "Loops in globular proteins: a novel category of secondary structure". Science. 234 (4778): 849–855. [4] Branden C. and Tooze J. "Introduction to Protein Structure" Garland Publishing, New York. 1990 and 1991. [5] Berg JM, Tymoczko JL, Stryer L (2002). "Section 3.5: Quaternary Structure: Polypeptide Chains Can Assemble Into Multisubunit Structures". Biochemistry (5. ed., 4. print. ed.). New York, NY [u.a.]: W. H. Freeman. [6] Veverka V et al. (2009). Characterization of the structural features and interactions of Sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation. The Journal of biological chemistry. 284. 10890-900. [7] Mallikaratchy P (2017). "Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens". Molecules. 22 (2): 215. [8] Lakhin, A. V., Tarantul, V. Z., & Gening, L. V. (2013). Aptamers: problems, solutions and prospects. Acta naturae, 5(4), 34–43.
