Designing Insulin Analogues with Lower Binding Affinity to Insulin Receptor than That of Insulin Icodec

1. Introduction

Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia, which remains a global health challenge with significant morbidity and mortality [1]. Insulin therapy stands as a cornerstone in the management of diabetes, yet the quest for optimized insulin analogues keeps driving continued innovation in therapeutic development for diabetic patients [2]. The evolution from conventional insulin formulations to engineered analogues has been propelled by the pursuit of achieving enhanced pharmacokinetic profiles, notably extended duration of action coupled with reduced risk of hypoglycemia [3]. For instance, insulin icodec of Novo Nordisk is a long-acting insulin analogue for better management of blood sugar levels in people with diabetes [4,5,6]. Insulin icodec is designed to provide a steady release of insulin throughout the day, mimicking the natural insulin production in the body [7,8,9]. Insulin icodec is typically administered through injection once a week, which helps lower blood sugar levels by allowing glucose to enter the body’s cells, where it is subsequently used for energy production [10,11]. Moreover, insulin icodec has a distinct pharmacokinetic profile compared to other long-acting insulin analogs. It exhibits a long duration of action, with a half-life of approximately 196 hours, leading to improved glycemic control and reduced hypoglycemia risk [12,13,14].

Interestingly, Icosema (of Novo Nordisk, too) [15,16,17,18] represents a combination medication that consists of insulin icodec and semaglutide, which belongs to a class of medications called glucagon-like peptide-1 (GLP-1) receptor agonists and helps regulate blood sugar levels by stimulating the release of insulin, reducing the production of glucagon (a hormone that increases blood sugar levels), and slowing down the digestion process for weight reduction [19,20,21,22].

Figure 1. A brief illustration of the two-dimensional structures of native human insulin and insulin icodec [3,23,24]. In this figure, the amino acid residues with pink backgrounds represents the positions of the three site-specific mutations [25,26] (Y14A_E, Y37B_H, F46B_H) of insulin icodec.

Figure 1. A brief illustration of the two-dimensional structures of native human insulin and insulin icodec [3,23,24]. In this figure, the amino acid residues with pink backgrounds represents the positions of the three site-specific mutations [25,26] (Y14A_E, Y37B_H, F46B_H) of insulin icodec.

In light of the two-dimensional structures of native human insulin and insulin icodec as shown in Figure 1, below is a list of structural modifications of insulin icodec compared to native human insulin [27,28,29]:

• addition of a fatty acid (K50B_C20, Figure 1 and Figure 2);
• deletion of threonine at position B30 (T51B_del, Figure 1 and Figure 2);
• a site-specific missense mutation, Y14A_E (Figure 1 and Figure 2);
• a site-specific missense mutation, Y37B_H (Figure 1 and Figure 2);
• a site-specific missense mutation, F46B_H (Figure 1 and Figure 2).

Overall, these structural modifications (Figure 2) in insulin icodec result in a more stable and longer-acting insulin analogue compared to regular insulin, providing a more consistent and sustained blood glucose-lowering effect:

• insulin icodec is able to form aggregates or clusters at the subcutaneous injection site, gradually releasing into the bloodstream over an extended period.
• insulin icodec undergoes structural modifications that increases its stability and solubility and preventing enzyme-mediated degradation and rapid clearance [34].
• insulin icodec is conjugated with a fatty acid at position B30 (K50B_C20, Figure 2). After injection, the fatty acid chain in insulin icodec interacts with albumin in the subcutaneous tissue, forming reversible albumin-insulin complexes. These complexes act as a reservoir, gradually releasing insulin icodec into the bloodstream, increasing its fat solubility and allowing it to bind to fatty acid-binding proteins, forming a depot of the insulin icodec reversibly bound to albumin.
• the incorporation of fatty acid chains facilitate the formation of stable hexameric structures, thereby delaying insulin absorption and promoting sustained release.
• with respect to the addition of a fatty acid (K50B_C20, Figure 2), it is conceivable that the deletion of threonine at position B30 (T51B_del, Figure 2) is for K50B_C20 to take place easier and more efficiently than without the deletion of threonine at position B30.
• two missense mutations of insulin icodec (Y37B_H and F46B_H) contributed to its prolonged duration of action through the induction of a modest decrease in the binding affinity of insulin icodec and its receptor (IR) [14]. Specifically, it is entirely due to the site-directed mutation Y37B_H that the salt bridge at the binding interface between insulin icodec and IR becomes weaker (from 3.204 Å to 3.669 Å), but is still not disrupted by the site-directed mutation Y37B_H, such that the binding affinity of ligand-receptor is lowered but not eliminated by the site-directed mutation Y37B_H [14], and thereby ensuring downstream signal transduction for the prolonged blood glucose-lowering effect of insulin icodec.

2. Motivation

As reported for the first time in [14], two missense mutations of insulin icodec (Y37B_H and F46B_H) contributed to its prolonged duration of action of insulin icodec through the induction of a modest decrease in the binding affinity of insulin icodec and its receptor [14], offering improved glycemic control and reduce treatment burden, thus enhancing patient adherence and overall outcomes in diabetes management [35,36]. The motivation behind this study stems from the need to exhaustively explore the entire insulin-IR molecular space [37,38,39,40] and to keep pushing forward the biophysical limit(s) of the binding affinity between insulin analogues and its receptor to finally reach a balance between the pharmacokinetic and therapeutic limits of structurally conceivable insulin analogues for improved duration of action and enhanced control of blood glucose levels for diabetic patients in future.

3. Materials and Methods

As listed in Table 1, as of 2024/05/07 14:57:32, there are a variety of experimental complex structures of insulin (analogues) bound to its receptor (IR), such as PDB entries 7PG3 (insulin receptor bound to 3 insulins), 7PG4 (insulin receptor bound to 2 insulins), 6SOF (insulin receptor bound to 4 insulins).

Among the insulin receptor-related structures listed in Table 1, PDB entry 6SOF (Figure 3) [44] is the only experimental complex structure of insulin bound to IR, where all four distinct binding sites of the IR dimer are saturated by four insulin molecules. Therefore, PDB entry 6SOF [44] is chosen here as the structural template for subsequent structural modelling of insulin icodec bound to IR.

Briefly, the amino acid sequences of the ten chains of native human insulin and IR (according to PDB entry 6SOF [44] are listed in italics in fasta format as below,

>6SOF_1|Chains A|Insulin receptor|Homo sapiens (9606)

HLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCLKGLKLPSRTWSPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQILKELEESSFRKTFEDYLHNVVFVPRPS

>6SOF_2|Chains B|Insulin receptor|Homo sapiens (9606)

HRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPEERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLIVLYEVSYRRYGDEELHLCVSRKHFALERGCRLRGLSPGNYSVRIRATSLAGNGSWTEPTYFYVTDYLDVPSNIAK

>6SOF_1|Chains C|Insulin receptor|Homo sapiens (9606)

HLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCLKGLKLPSRTWSPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQILKELEESSFRKTFEDYLHNVVFVPRPS

>6SOF_2|Chains D|Insulin receptor|Homo sapiens (9606)

HRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPEERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLIVLYEVSYRRYGDEELHLCVSRKHFALERGCRLRGLSPGNYSVRIRATSLAGNGSWTEPTYFYVTDYLDVPSNIAK

>6SOF_3|Chains E|Insulin|Homo sapiens (9606)

GIVEQCCTSICSLYQLENYCN

>6SOF_4|Chains F|Insulin|Homo sapiens (9606)

FVNQHLCGSHLVEALYLVCGERGFFYTPKT

>6SOF_3|Chains G|Insulin|Homo sapiens (9606)

GIVEQCCTSICSLYQLENYCN

>6SOF_4|Chains H|Insulin|Homo sapiens (9606)

FVNQHLCGSHLVEALYLVCGERGFFYTPKT

>6SOF_3|Chains I|Insulin|Homo sapiens (9606)

GIVEQCCTSICSLYQLENYCN

>6SOF_4|Chains J|Insulin|Homo sapiens (9606)

FVNQHLCGSHLVEALYLVCGERGFFYTPKT

>6SOF_3|Chains K|Insulin|Homo sapiens (9606)

GIVEQCCTSICSLYQLENYCN

>6SOF_4|Chains L|Insulin|Homo sapiens (9606)

FVNQHLCGSHLVEALYLVCGERGFFYTPKT

In the only experimental complex structure of four insulins bound to IR (PDB entry 6SOF (Figure 3) [44]), the first IR molecule is defined as >6SOF_1|Chains A and >6SOF_2|Chains B, the second IR molecule is defined as >6SOF_1|Chains C and >6SOF_2|Chains D, the first insulin molecule is defined as >6SOF_3|Chains E and >6SOF_4|Chains F, the second insulin molecule is defined as >6SOF_3|Chains G and >6SOF_4|Chains H, the third insulin molecule is defined as >6SOF_3|Chains I and >6SOF_4|Chains J and the fourth insulin molecule is defined as >6SOF_3|Chains K and >6SOF_4|Chains L, respectively.

Subsequently, a huge ($s=g(51,3)=\frac{51!}{3!(51-3)!}\times {20}^k$ [38]) set of insulin analogues were generated with in-house Python script with three site-specific missense mutations introduced into native insulin sequences. Afterwards, homology structural modeling was carried out using Modeller [46] with PDB entry 6SOF [44] as the structural template. Finally, the binding affinity between insulin analogues and IR was calculated using Prodigy [47,48] for native insulin (10000 times), insulin icodec (10000 times) and also for $s=g(51,3)=\frac{51!}{3!(51-3)!}\times {20}^k$ [38] insulin analogues (1 time).

4. Results

As experimentally determined in human insulin receptor ectodomain bound by 4 insulin (PDB entry 6SOF [44,49]) using a combined approach of cryo-EM and atomistic molecular dynamics simulation, the structure of the entire dimeric insulin receptor ectodomain is saturated with four insulin molecules, i.e., the first IR molecule (abbreviated as A in Table 2, Table 3, Table 4 and Table 5), the second IR molecule (abbreviated as C in Table 2, Table 3, Table 4 and Table 5), the first insulin molecule (abbreviated as E in Table 2, Table 3, Table 4 and Table 5) consisting of >6SOF_3|Chains E and >6SOF_4|Chains F, the second insulin molecule (abbreviated as G in Table 2, Table 3, Table 4 and Table 5) consisting of >6SOF_3|Chains G and >6SOF_4|Chains H, the third insulin molecule (abbreviated as I in Table 2, Table 3, Table 4 and Table 5) consisting of >6SOF_3|Chains I and >6SOF_4|Chains J and the fourth insulin molecule (abbreviated as K in Table 2, Table 3, Table 4 and Table 5) consisting of >6SOF_3|Chains K and >6SOF_4|Chains L, respectively. Therefore, for each insulin analogue with one set of missense mutations, there are a total of eight values of intermolecular binding affinity (K_d) to be calculated by Prodigy [47,48], as listed in Table 2, Table 3, Table 4 and Table 5.

In Table 3, Table 4 and Table 5, to increase the likelihood of the insulin analogues designed here possessing an extended duration of action than insulin icodec, all $s=g(51,3)=\frac{51!}{3!(51-3)!}\times {20}^k$ [38] insulin analogues were computationally screened with a collection of criteria as below:

• the Prodigy-calculated K_d of the insulin analogue to its receptor (AE) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the first engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.
• the Prodigy-calculated K_d of the insulin analogue to its receptor (AG) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the second engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.
• the Prodigy-calculated K_d of the insulin analogue to its receptor (AI) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the third engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.
• the Prodigy-calculated K_d of the insulin analogue to its receptor (AK) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the fourth engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.
• the Prodigy-calculated K_d of the insulin analogue to its receptor (CE) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the first engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.
• the Prodigy-calculated K_d of the insulin analogue to its receptor (CG) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the second engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.
• the Prodigy-calculated K_d of the insulin analogue to its receptor (CI) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the third engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.
• the Prodigy-calculated K_d of the insulin analogue to its receptor (CK) is larger than that of native insulin or insulin icodec or both, i.e., the binding affinity of the fourth engineered insulin analogue to its receptor is lower that that of native insulin or insulin icodec or both.

5. Conclusion and Discussion

For the first time, through a comprehensive structural and biophysical analysis [50] of the insulin (both native and icodec) structures bound to its receptor, this article puts forward a a set of insulin analogues with lower binding affinity to insulin receptor than that of insulin icodec from a structural and biophysical point of view [51]. Overall, this article calls for subsequent in vitro and in vivo evaluations of the efficacy and prolonged action of the engineered insulin analogues to pharmacokinetically test whether these analogues acutally surpass that of insulin icodec as hopeful candidates for next-generation insulin analogue therapies with improved duration of action [14,52] and enhanced control of blood glucose levels for diabetic patients in future.

The development of insulin analogues with extended duration of action while maintaining therapeutic efficacy represents a significant advancement in diabetes management [14]. With computational modeling and structure-based sequence design, this study leveraged insights from protein engineering and molecular biophysics to guide the rational design of insulin analogues with potentially prolonged duration of action compared to insulin icodec, as evidenced by the structural biophysical analysis as listed in Table 2, Table 3, Table 4 and Table 5.

Furthermore, while this study represents another step towards the development of next-generation insulin therapies with improved duration of action, the entire process of the design of insulin analogues with lower binding affinity to insulin receptor than that of insulin icodec, along with the structural biophysics-based strategy for the molecular design, is essentially also a process of the construction of a insulin-IR based mini general intermolecular binding affinity calculator [22,37,38] based on the experimentally determined human insulin receptor ectodomain bound by 4 insulin (PDB entry 6SOF).

6. Ethical Statement

No ethical approval is required.

7. Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this work, the author used OpenAI’s ChatGPT in order to improve the readability of the manuscript, and to make it as concise and short as possible. After using this tool, the author reviewed and edited the content as needed and takes full responsibility for the content of the publication.