Native Design of Soluble, Aggregation-Resistant Bioactive Peptides

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Native design of soluble, aggregation-resistant bioactive peptides: Chemical evolution of human glucagon Piotr A. Mroz, Diego Perez-Tilve, Fa Liu, John P. Mayer, and Richard D. DiMarchi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00923 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Native Design of soluble, aggregation-resistant bioactive peptides: Chemical evolution of human glucagon

Piotr A. Mroz1, Diego Perez-Tilve 2, Fa Liu 3, John P. Mayer*3, and Richard D. DiMarchi*1,3

1

Department of Chemistry, Indiana University, Bloomington, IN 47405 USA. 2Department of

Medicine, Metabolic Diseases Institute, University of Cincinnati, Cincinnati, OH, 45267 USA. 3

Novo Nordisk Research Center, Indianapolis, IN 46241.

ABSTRACT Peptide-based therapeutics commonly suffer from biophysical properties that compromise pharmacology and medicinal use. Structural optimization of the primary sequence is the usual route to address such challenges while trying to maintain as much native character, and avoiding introduction of any foreign element that might evoke an immunological response. Glucagon serves a seminal physiological role in buffering against hypoglycemia, but its low aqueous solubility, chemical instability and propensity to self-aggregate severely complicate its medicinal use. Selective amide bond replacement with metastable ester bonds is a preferred approach to the preparation of peptides with biophysical properties that otherwise inhibit synthesis. We have recruited such chemistry in the design and development of unique glucagon prodrugs that have physical properties suitable for medicinal use and yet rapidly convert to native hormone upon exposure to slightly alkaline pH. These prodrugs demonstrate in vitro and in vivo pharmacology when formulated in physiological buffers that is nearly identical to native hormone when solubilized in conventional dilute hydrochloric acid. This approach provides the best of both worlds, where the pro-drug delivers chemical properties supportive of aqueous formulation and the native biological properties.


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INTRODUCTION Peptides constitute an indispensable class of medicinal agents whose development is frequently compromised by chemical and physical properties which adversely impact their integrity.1,2 In extreme cases this can result in conversion of a native, unconstrained secondary structure to a toxic non-native species characterized by insoluble amyloid-like fibrils.3,4 Such challenges are typically addressed by highly optimized, delicate formulations5 or change to the primary sequence.6 While structural refinement is a hallmark of medicinal chemistry it entails careful balancing of chemical and biological properties guided by exhaustive assessment of efficacy and safety. Insulin is a classic example of a peptide whose physical character and bioactivity can be destroyed by vigorous agitation and even modest temperature change.7,8 Insulin lispro is an analog derived by inversion of two native amino acids that serves to appreciably reduce self-association.9 This subtle structural change enables an accelerated pharmacokinetic profile, greater resistance to fibrillation to render it superior to native insulin in prandial and pump-infused insulin therapy.10,11 Glucagon (1) is a 29-residue pancreatic hormone secreted in response to low blood glucose and as such represents the primary medication in treatment of life-threatening hypoglycemia.12,13 While manufacture of the peptide has advanced in the last fifty years from pancreatic glandular extraction to rDNA biosynthesis14,15, its medicinal use requires reconstitution in dilute hydrochloric acid immediately prior to injection.16 Glucagon chemically degrades at low pH via a mid-sequence aspartimide formation and deamidation of multiple amides.17,18 Advancements in development of soluble formulation of native glucagon using aqueous5,19 and non-aqueous formulations20 have been recently reported. We on the other hand have promoted chemical modifications in the native sequence to dramatically improve physical properties.21-25 Nonetheless, the safety concerns, particularly immunogenicity that are inherent to any structural change constitute a persistent uncertainty.26 An ideal candidate would provide native biological function, but be chemically fortified for the period prior to administration.



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2

a

(1)

5

7 8

11

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16

29

HS QGT FT S DYS KYLDS RRAQDFVQWLMNT

b

c

Figure 1. (a) Human glucagon amino acid sequence highlighting in red seven hydroxylated amino acids (serine and threonine). (b) Conceptual design of depsi-peptide where a single metastable ester inactivates the hormone, but provides much enhanced aqueous solubility in physiological buffers. R1 can represent a side chain of an amino acid preceding a native serine or threonine and R2 = H or CH3. (c) Conceptual design of an enzymatically triggered glucagon depsi-peptide where a single metastable ester is protected from premature O-to-N acyl shift by a chemical modification that is susceptible to rapid removal upon in vivo application. R1 and R2 as defined in (a) and R3 = protease substrate.


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The use of O-acyl isopeptides, championed by Kiso and co-workers is a well-established technique for the synthesis poorly soluble, aggregation-prone peptides.27-29 Once chemically assembled and purified under acidic conditions, such depsi-peptides undergo an O-to-N acyl shift at alkaline pH to generate native amide sequence. This strategy has been successfully applied to notoriously difficult peptides such as Aβ(1-42)30, islet amyloid polypeptide31, as well human insulin32, enzyme prodrugs33 and “switch peptides” designed to study conformational transitions of amyloid structures.34-36 The speed of this transformation is of modest consequence when used for synthetic purposes but it is of vital importance as we envision its therapeutic use, as a matter of minutes can be the difference between life and death in rescue from excessively low blood glucose. Conversely, if the depsi-bond is insufficiently stable it will prematurely rearrange, or hydrolytically degrade in aqueous formulations before therapeutic use. Consequently, glucagon represents an ultimate challenge of whether depsi-bond pro-drugs can be designed to convert within minutes at physiological pH to native hormone, and yet possess sufficient solubility and stability as an aqueous, ready to use formulation. Of the seven alcohols in the glucagon sequence four are serine and three are threonine residues. Five of these seven amino acids are positioned in the midsection of the peptide [Figure 1a]. We envisioned that the introduction of a single O-acyl isopeptide unit would significantly improve aqueous solubility of the analog and enable conversion to the native backbone at physiological pH [Figure 1b]. Our results illustrate that serine and threonine-based depsi-peptides

readily

convert

in

vivo

to

generate

pharmacology

that

is

virtually

indistinguishable from the native hormone. Furthermore, the ester bond can be stabilized by judicious selection of chemical sequence and position such that a slightly acidic aqueous solution can be formulated and stored for extended periods. We also report that enzymaticallylabile protection of the depsi-peptide alpha-amine provides much enhanced stability for those formulations requiring extended or higher temperature stability, without any apparent change in pharmacodynamic action. This chemical-biotechnology exemplifies the application of classical



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chemistry to evolve the performance of glucagon as a life-saving drug, and is conceivably applicable to other peptides and proteins.

RESULTS & DISCUSSION A family of glucagon analogs possessing a single depsi-peptide bond was synthesized by automated Fmoc/tBu, or a combination of Boc/Bzl and Fmoc/Bzl solid-phase peptide chemistry [Table 1, Table S1 and Figure S2]. The depsi-bond in analogs 2-5, 8-12 and 16-17 was formed manually by coupling the subsequent Fmoc-protected amino acid with DIC and catalytic DMAP for 12 to 24 hours. In analogs 6-7 and 13-15 the isoacyl dipeptide BocThr[Fmoc-Phe]-OH or Boc-Thr[Fmoc-Asp(OtBu)]-OH was used to ensure enantiomerically pure depsi constructs.37 (Details in Supporting Information) Peptide

Structural Modifications

Abbreviation

2

#1: D15-depsi-S16

D15:S16

3

#2: T16

D15:T16

4

#1: F6-depsi-T7

F6:T7

5

#1: G4-depsi-T5

G4:T5

6

#3: F15

F15:T16

7

#4: D6

D6:T7

8

#2: T16(Ac)

D15:T16(Ac)

9

#5: Aib2,T5(Ac)-amide

B2,G4:T5(Ac)-am

10

#5: T5(K-Aze)

G4:T5(KZ)

11

#5: T5(K-P)

G4:T5(KP)

12

#5: T5(E-Aze)

G4:T5(EZ)

13

#4: T7(K-Aze)

F6:T7(KZ)

14

#4: T7(A-P)

F6:T7(AP)

15

#4: T7(E-P)

F6:T7(EP)

16

#4: T5(R)

G4:T5(R)

17

#4: T5(pE)

G4:T5(pE)

Table 1. Glucagon depsi-peptide analogs. (Gcg = Glucagon, Aze = Azetidine, pE = pyroglutamate; B = Aib, Z = Azetidine)


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The O-to-N acyl shift is a well characterized, pH sensitive reaction that has been appreciably used to enable the synthesis of peptides that prove difficult to prepare by conventional methods.27-29 To successfully employ this chemistry as a central ingredient to design of a glucagon analog intended for emergency treatment of hypoglycemia it is imperative that the conversion occurs nearly instantaneously. Consequently, as a first step we investigated the O-to-N acyl conversion of depsi-glucagon analogs to biologically active hormone under physiological conditions (PBS pH 7.4 and 37 oC) [Figure 2 and S3-4]. Three positions in the peptide sequence

Figure 2. Kinetics of O-to-N acyl shift for depsi-glucagon analogs 2-7, in PBS pH 7.4 at 37 oC. Each line represents the fitting of the observed disappearance of each depsi-peptide over time to first order reaction kinetics. ([A] –area under the HPLC peak, UV, λ = 214 nm)

were assessed (T5, T7, S16), and a single comparison of serine vs threonine at positon 16. Analysis was performed by quantitative LC-MS, as timed aliquots were quenched by dilution in ice-cold, 1% TFA. There was a nearly fiftyfold difference in the dynamic reaction rate as peptide



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5 [G4:T5] proved fastest with a recorded half-time of ~13 seconds and analog 2 [D15:S16] being slowest at 559 seconds [Table 2]. The nature of the hydroxylated amino acid at the depsibond substantially affected the reaction rate. This is clearly demonstrated when comparing peptide 3 with threonine at position 16 against the comparable analog 2 that differs only with serine at the same position. The presence of a methyl group at the side-chain beta-carbon of threonine accelerated the reaction by approximately fivefold to a half-time of slightly less than two minutes (~111 seconds).38 Furthermore, the specific location of the threonine depsi-bond exerted an even greater influence on the conversion rate than this single change from serine to threonine at position 16. The respective rates for T5, T7 and T16 were approximately 13, 29 and 111 seconds, which represents an 8.5x range difference in relative speed. This rate of conversion is consistent with the application of this chemistry to a glucagon pro-drug intended for emergency use. Of equal importance was the dynamic range as it suggested that the peptide stability could be fine-tuned through subtle changes in location of the depsi-bond and the choice of serine or threonine.

o

Peptide

t ½ (pH 7, 37 C) [seconds]

t ½ (pH 5, RT) [hours]

2

559.0

118.9

3

110.5

25.7

4

29.2

2.1

5

12.7

0.5

6

56.3

n.d.

7

52.4

n.d.

Table 2. Determined half-time in the O-to-N acyl shift reaction.

The differences in reaction rate between the three threonine analogs 3, 4, 5 may reflect dissimilarity in the nature of the specific depsi-dipeptides, or secondary structural differences in


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the N-terminal half of glucagon. Analogs 6 and 7 were prepared to investigate this question as they constitute single site changes to peptides 3 and 4. In peptide 7 the depsi-bond is located at the 6-7 position in analogous fashion to peptide 4, but phenylalanine 6 is replaced with aspartic acid, the amino acid native to position 15. The rate of reaction is slowed nearly twofold [Table 2]. The opposite effect was observed in analog 6 where aspartic acid 15 was replaced with phenylalanine and the reaction rate nearly doubled, relative to 3. These results collectively suggest that the difference in the speed of reaction for 3 and 4 can be explained by the nature of the dipeptides at positions 6-7 and 15-16, without any appreciable contribution from secondary structure. The half-life of the O-to-N acyl shift of one minute or less satisfies the first criteria in a design of glucagon pro-drug. However, this conversion also represents a point of chemical instability that needs to be suppressed in a pre-formulated drug prior to its administration. Consequently, the degree to which the reaction is slowed by changes in pH and temperature needed to be determined. Peptides 2 and 5 represent the extremes in the rate of reaction at physiological conditions and their conversion to biologically active native glucagon was observed to be significantly reduced when studied at pH 5 and room temperature [Figures S5S7]. Where peptide 5 converted with a half-life of ~13 seconds at physiological conditions it took thirty minutes when the pH and temperature were subtly reduced, a decrease of 140x [Table 2]. Peptide 2 exhibited an even greater relative response to change in temperature and pH as the rate of reaction slowed by more than 750x, now requiring five days for half conversion to native glucagon. While these changes represent dramatic differences in the speed of chemical reaction they also illustrate that such a depsi-peptide would require additional fortification in its stability for conventional use as a pre-formulated medicine. The primary purpose of the depsi-bond is to render native glucagon sufficiently soluble in aqueous solution such that it could be used immediately as a pre-formulated drug at time of emergency use. The O-to-N acyl shift complicates the determination of inherent aqueous



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solubility for a depsi-peptide. This can be approximated by inhibiting the intramolecular rearrangement by acetylation in the alpha-amine of the depsi-bond, thus rendering it inert and the peptide suitable for biophysical study. Peptides 8 and 9 represent acetylated analogs of peptides 3 and 5. Peptide 8 [D15:T16(Ac)] is stable in PBS demonstrating the importance of the single alpha-amine in the conversion of depsi-peptide 2 to native glucagon. Its solubility in PBS is in excess of 2.5 mg/mL, which is more than tenfold relative to native glucagon (1) and sufficient to support formulation at 1 mg/mL [Figure S10]. The aqueous solubility of the closely related peptide 9 [G4:T5(Ac)-am] where the depsi-bond is positioned at residues 4 and 5 is also sizably enhanced relative to glucagon, but less so than analog 8. These results demonstrate that a single depsi-bond can provide sufficient enhancement in aqueous solubility to constitute a concentration that would support medicinal use, without excessive change in the volume administered. Furthermore, the specific location of the depsi-bond exerts differential effects, with the mid-sequence placement providing enhanced solubility. Peptides 8 and 9 were also used to evaluate the stability of the glucagon backbone, including hydrolytic cleavage of the single ester bond in PBS (pH 7.4, room temperature). The native sequence proved relatively inert under these conditions with the depsi-bond as expected being the most sensitive degradation point. Analog 8 demonstrated good stability through nearly a week of incubation, while in comparison the single depsi-bond in analog 9 resulted in almost 20% degradation [Figure 3a]. To further test the inherent stability of peptide 8 it was incubated at elevated temperature in a slightly modified physiological buffer of citric acid/phosphate (pH 7 at 40 oC) for five days. It exhibited a level of depsi-bond hydrolysis that was less than 9 when the latter was incubated at room temperature, [Figure S11 and 3a]. The difference in stability to general aqueous hydrolysis reflects the propensity in O-to-N acyl rearrangement, which is a measure of the inherent stability of the ester bond. This positional difference in stability is an important consideration in the design of a depsipeptide that meets all of the necessary criteria for medicinal application; speed to biological action, sufficient solubility and chemical stability as a prodrug in aqueous solution.


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a

b

Figure 3 (a) Peptide stability as a function of incubation time in PBS at room temperature. Peptides 8 and 9 are characterized in Table 1 as D15:T16(Ac) and B2,G4:T5(Ac)-am. The latest eluting peak in 9 chromatograms represents hydrolysis of the depsi-peptide. [* represents the initial depsi-peptide]. (b) Stability of glucagon depsi-peptide analog 10 in citric acid/disodium phosphate buffer after 5 day incubation at 4 oC and room temperature. [* represent starting depsi peptide]



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To further enhance the chemical stability of the depsi-peptide without compromising the rate at which it can generate active hormone a reversible means of protecting this ester bond was explored. Further lowering of pH in the formulated solution was envisioned as being undesirable as it would lead to increased acid-mediated hydrolytic degradation of the depsibond and other native amino acids, especially amides.19,20 Similarly, lowering the storage temperature to refrigerated conditions is unacceptable as an emergency use product should not be compromised by such a restriction. The acetylation of the depsi-peptide alpha-amine provides a sufficient increase in chemical stability without compromising aqueous solubility, but it is incapable of rearrangement to active hormone. The prospect of using an enzymaticallylabile protection is attractive as it masks the nucleophile until that point when it is liberated by proteolysis [Figure 1c].34-36 The primary challenges to such a design pertain to the speed and the certainty in which the nucleophile is generated as in its absence there will be no biological activity. Several candidate substrates were considered and a select number were assembled (10-17), across two different locations in the glucagon sequence. An initial set of depsi-containing analogs was prepared in the G4:T5 sequence (peptides 10-12). The previous stability studies indicated this depsi-bond to be least stable and as such it provides the more stringent test for evaluating the integrity of the protection with putative, enzymatically susceptible substrates. Each of these three analogs (10-12) would be expected to generate native glucagon once cleaved by an enzyme, followed by an O-to-N acyl shift. This sequence site also constitutes a more straightforward synthetic target since the glycine at position four can be esterified to the threonine at residue five without fear of loss in stereochemical purity. The threonine at position 5 was incorporated as Thr(Trt) and the trityl protecting group was easily removed by treatment with 1% TFA. Esterification with Fmoc-Gly provided enantiomerically pure depsi-peptide which was further extended with the N-terminal glucagon tripeptide in standard Fmoc fashion [Figure S12a].


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Glucagon analog 10 [G4:T5(KZ)] represents a depsi-peptide that has been designed to rapidly cleave to 5 when exposed to the enzyme DPPIV. This exopeptidase is ubiquitous and demonstrates high substrate preference for dipeptides that include penultimate cyclic amino acids, such as proline.39 The dipeptide lysine-2-carboxy-azetidine is an extremely favorable substrate that demonstrates a low Km and high kcat.40 Peptide 10 demonstrates excellent solubility (>2 mg/mL) in a variety of aqueous physiological buffers [Figure S13]. Relative differences in solubility appear to be a function of buffer salt concentration and specific pH. It should be noted that the additional lysine in the substrate dipeptide increases the cationic nature of the peptide relative to native glucagon and this also assists aqueous solubility in the physiological pH range.23 The greatest concentration of 10, (> 3 mg/mL) was achieved at pH of 7 in an aqueous buffer composed of 0.1M citric acid and 0.2M disodium phosphate (room temperature). This is 3x the concentration of native glucagon administered to patients once solubilized in dilute hydrochloric acid. Comparative solubility of analogs 10-15 was studied in PBS pH 7.4, at room temperature [Figure 4]. All analogs are of much higher solubility then native glucagon, and the location of the depsi-bond and the specific nature of the added substrate subtly, but meaningfully influence the result. The solution stability of 8 and 10 was assessed to determine whether there might be subtle differences imposed by simple acetylation relative to an extended dipeptide that terminates with a free alpha-amine. Both peptides were formulated in citric acid, phosphate buffer in a pH range of pH 3-7, and incubated for extended time at three different temperatures (4, 23, and 40 oC). The stability was determined with daily sampling and analysis by LC-MS. In Figure 3b the HPLC analysis of 10 is shown after five days incubation at pH 5, 6 and 7. At 4 oC the peptide shows good stability in each of the three buffers, without slight apparent preference for lower pH. There is a variable degree of degradation observed at pH 7 (5 day), with complete loss at 40 oC and a fractional amount at 23 oC [Figure S14]. The primary basis of chemical



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degradation was determined to be the hydrolytic cleavage of the ester bond to generate two peptide fragments.

Figure 4. Solubility of the glucagon and depsi-peptides 10-15 in PBS pH 7.4, at room temperature. [doted black line represent pharmaceutically relevant concentration level for use of emergency glucagon treatment]

At pH 6, peptide 10 appeared comparably stable at 4 oC and room temperature to pH 7 buffer at refrigerated temperature, [Figure 3b]. At 40 oC there was finite degradation (~40%) but much less than was observed at the elevated temperature and pH of 7. The analyses of 10 when incubated at pH 5 showed minor hydrolysis of the depsi-bond, and only when the temperature was elevated to 40 oC. Biophysical stability of the depsi-analogs in comparison to native glucagon (1) was investigated at pH 3 and 4 due to extremely low solubility of 1 under physiological conditions. Formation of amyloid-like fibrils was measured by thioflavin-T mediated fluorescence [Figure S15].41-43 As expected the aggregation of native glucagon (1) and depsi analogs 8 and 10 stored at 4 oC was minimal, in this time period. At elevated temperatures (23 and 40 oC) 1 exhibited a


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strong florescence, indicative of biophysical aggregation. Peptides 8 and 10 show no evidence of such aggregation at 23 oC, and only a trace at 40 oC. The collective results demonstrate that the depsi-peptide bond G4:T5 once extended with a specific dipeptide (KZ) possesses high aqueous solubility at pH 5, and sizably enhanced chemical stability without propensity to form amyloid-like fibrils. While the prospect for cytotoxity of the soluble non-aggregating depsipeptides is low, confirmation would need to be rigorously evaluated as part of a toxicology package for any clinical development candidate. Further comparative analysis of extended depsi-glucagon analogs (10-15) incubated in PBS at room temperature revealed no major difference in ester bond stability between the investigated peptides [Figures S16 and S17]. Consequently, it seemed plausible to proceed with in vivo study to determine whether protease cleavage and subsequently O-to-N rearrangement will occur to provide a rapid increase in blood glucose, comparable to native glucagon solubilized in hydrochloric acid.

Figure 5. The in vitro activity of glucagon depsi –peptides 5, 8, 9, 15 relative to native glucagon. The measured activity is an indirect function of cAMP synthesis as assessed in an engineered cell assay where the human glucagon receptor is over-expressed and coupled to a luminescence reporter.



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The assessment of in vitro potency at the glucagon receptor was conducted using an engineered cellular assay that measured cAMP production. The depsi-peptide 5 was observed to be equipotent to the glucagon standard, which was presumed to be a reflection of the rapid conversion to native sequence under the conditions of biochemical study [Figure 5]. Analogs 8 and 9 represent acetylated, depsi-peptides differing in the location of the ester bond and they demonstrate activity of less than 1% of native glucagon. This validates that the insertion of a single ester bond is highly destructive to receptor signaling and subsequent biochemical action. Interestingly, the depsi-peptides 10 and 15 while reduced in potency 20-50x relative to glucagon (1) and peptide 5, were ~10x more potent than the comparably acetylated analogs 8 and 9. A plausible, but unconfirmed basis for the increased potency suggests a low level surface bound proteolysis of the dipeptide substrate to enable rearrangement to active hormone. The biological activity of select depsi-peptide analogs was evaluated in non-diabetic rats. Peptides 1, 2, 3 and 5 were formulated in 0.01N HCl immediately prior to administration at a 10 nmol/kg dose. Elevation in blood glucose was followed through a two-hour period and each of the peptides exhibited a statistically significant increase separable from vehicle control, but without difference from one another [Figure 6a and S18a]. The pharmacodynamic response is consistent with historical glucagon action, peaking within 30 min and returning to initial glucose level at two hours.44 These results corroborate the rapid chemical conversion from an inactive precursor to a fully active hormone, as previously demonstrated by chromatography [Figure S4] and biochemical assay [Figure 5]. The absence of any biological difference across these three depsi-peptides was unexpected, as the in vitro rate in chemical rearrangement varies between one to eleven minutes [Table 2]. It is possible that a lower dose might demonstrate some subtle difference, but it is well-recognized that emergency glucagon therapy is administered at an excessively high dose to ensure full action. In a subsequent test of in vivo action peptide 10 formulated in PBS was compared to 1 and 5 solubilized in dilute hydrochloric acid [Figure 6b and S18b]. The three peptide treatments


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a

b

c

Figure 6. (a) Change in blood glucose level in non-diabetic rats after administration of native glucagon (1), depsi-peptides 2, 3 and 5 [all formulated in 0.01N HCl; dose: 10 nmol/kg]; (b) Change in blood glucose level in non-diabetic rats after administration of native glucagon (1), depsi-peptide 5 [formulated in 0.01N HCl] and 10 [formulated in 50 mM sodium phosphate with 150 mM sodium chloride pH 7.4; dose: 10 nmol/kg]. Vehicle (I) was 50 mM sodium phosphate with 150 mM sodium chloride pH 7.4 buffer and Vehicle (II) was 0.01N HCl. (c) Change in blood glucose level in normal rats after administration of depsi-peptides 15 at 10 nmol/kg [formulated in PBS] in the presence of an orally administrated DPPIV inhibitor - Sitagliptin at 3, 10 and 30 mg/kg [formulated in PBS and administrated by gavage].



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demonstrated an increase in blood glucose relative to vehicle that was nearly identical to the first study. The rise in glucose peaked within thirty minutes at a magnitude that was approximately 30 mg/dL higher than identical vehicle treatment. Once again the unprotected depsi-peptide 5, solubilized in dilute acid was identical to similarly administered glucagon. Most importantly, 10 was equally potent in vivo, despite the fact that it was no more than 3% the potency of the native hormone when studied in vitro [Figure 5]. Changing the relative position of depsi bond (G4:T5 vs F6:T7) as well the character of DPPIV substrate (KZ vs EP) was found to have no apparent effect on the biological activity of 13 [F6:T7(KZ)], 15 [F6:T7(EP)] and 4 [F6:T7] [Figure S19], with recorded potency comparable to native glucagon (1). The relative performance of peptide 10 [Figure 6b] and 13 [Figure S19] is extremely encouraging as it indicates that a DPPIV substrate is capable of being rapidly removed, and presumably through the action of DPPIV. Two additional putative substrates for endogenous exopeptidases were also studied. Peptides 16 and 17 were designed for proteolysis by an Nterminal cationic aminopeptidase and a pyroglutamase, respectively. The test of these two depsi-peptides alongside peptide 5 and 10 is presented in Figure S20. Peptides 5 and 10 perform in analogous fashion to what is reported in Figure 6b while 16 and 17 elicit very little activity, indicating the superiority of DPPIV substrates. A potential dilemma in the use of a DPPIV substrate is the prospect of attenuated glucagon activity should a patient be using a DPPIV inhibitor, such as sitagliptin. Consequently, we explored whether sitagliptin (ApexBio) at a dose of 3, 10 or 30 mg/kg given orally 30 min prior to injection of 10 nmol/kg of analog 15 [F6:T7(PE)] would impair its ability to increase blood glucose [Figure 6c and S21]. Treatment with the enzyme inhibitor alone had only an insignificant effect on blood glucose. All of the mice treated with 15 displayed a significant elevation in blood glucose while the presence of sitagliptin demonstrates a subtle dose response that reduces the effect by a small fractional amount that was nearly without consequence.


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Whether sitagliptin is simply ineffective in inhibition of this DPPIV substrate under these experimental conditions or alternatively whether the substrate is being processed by an alternative endogenous enzyme is not possible to determine from the initial study. To further explore this question, we tested the ability of sitagliptin to inhibit GLP-1 proteolytic loss in potency. Paradoxically, exendin-4 has been reported to increase blood glucose upon acute administration to normal rats.45 When we compared the ability of an Aib2, DPPIV protected analog to change blood glucose it was apparent that there was a dramatic difference in potency relative to native hormone [Figure S22]. The addition of sitagliptin in comparable fashion to how it was used with depsi-peptides [Figure 6c and S21] was highly effective in strengthening the biological effect, demonstrating its ability to inhibit endogenous DPPIV. These results support the prospect that a secondary enzyme is sufficient to rapidly initiate the conversion of peptides 10 [Figure 6b] and 13 [Figure S19], and provide high potency glucagon agonism.

CONCLUSIONS Peptides are rarely the preferred molecular scaffold for drug development. As a structural class their molecular size and hydrophilicity compromise transfer across cellular membranes, largely limiting their use to peripheral, cell surface receptors and parenteral administration. Extensive investment to identify small molecule surrogates has only rarely proven successful.46 In the biotechnology era protein-based therapeutics have proven more attractive as drug candidates, largely as a result of their greater molecular size that typically serves to enhance higher-order structure and extend duration of action. Nonetheless, peptides such as insulin, parathyroid hormone, calcitonin, and a host of others including glucagon deliver indispensable pharmacology. In each instance appreciable investment has been made to identify conditions where these fragile macromolecules can be formulated as a drug, but with strict directions on how these aqueous solutions must be managed. The result we report here validate that a depsi-peptide bond can be used to increase the aqueous solubility of an



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otherwise poorly soluble peptide. It builds upon a rich body of prior synthetic reports where this simple, elegant tool has proven a unique approach in the preparation of peptides that could not otherwise be obtained by conventional methods.31,32 This current work demonstrates that this same chemistry can be used to address a fundamental obstacle in biophysical properties that undermine peptide medicinal chemistry, and could equally be applied to proteins of poor physical behavior through integrated semisynthesis of biosynthetic proteins. Glucagon is a life-saving drug and its clinical use has not changed since its registration in the 1950s. The drug requires reconstitution through a complicated procedure at the time of impaired cognitive function due to severe hypoglycemia. This byzantine requirement is anchored by the poor aqueous solubility of the hormone, coupled to its low chemical and physical stability in acidic solution. How many lives have been lost as a result of poor macromolecular physical properties is impossible to know, but it is a significant number as the majority of insulin-dependent diabetics do not carry a glucagon emergency kit for use at time of need. Nature designs peptides and proteins for physiological use, and medicinal application while pharmacologically related is in many ways distinctly different. The replacement of a single native amide bond with an ester disrupts self-association to dramatically enhance aqueous solubility. It is our vision that such a fortified glucagon could anchor the development of a delivery system similar to a ready to use epinephrine pen, and separately constitute the missing therapeutic in “closed loop” insulin therapy. Whether such a chemical refinement as simple as a single metastable ester can be used as a stable drug product intermediate that rapidly reverts to native structure and biological function is the basis of this report. Glucagon represents a stringent test of whether a macromolecule can successfully achieve this delicate balance. The results clearly indicate that a single ester can sufficiently enhance solubility and rapidly convert to full biological activity when assessed by in vitro and in vivo methods. The specific chemical nature of the depsidipeptide was of primary importance to the speed of reaction, with threonine-based peptides


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proving exceptionally fast and suitable for emergency use. However, the need for rapid chemical rearrangement was observed to simultaneously impose an instability that is incompatible with pre-formulation for conventional use. In other therapeutic instances with other macromolecules, serine-based depsi-peptides might prove a preferred alternative given the inherently increased stability relative to threonine. Reversibly protecting this metastable ester was observed to be possible through the use of slightly lower pH and an enzymatically labile substrate. Three substrates directed at three different putative endogenous enzymes were explored, peptides 10-15, 16 and 17. Fortuitously, the protected depsi-peptides designed to be susceptible to DPPIV proteolysis worked extremely well, demonstrating endogenous deprotection and intramolecular rearrangement in a period of minutes. A deeper analysis of these DPPIV-substrate extended depsi-peptides suggests that a second protease is supplementing the activation of these peptides, or less likely that these peptides are exceptional DPPIV-substrates and largely immune to enzyme inhibition. This mechanistic point deserves additional study. It would need to be established clinically that accidental coincident use of a DPPIV inhibitor would not block glucagon action as we observed in normal rats. This specific issue is not relevant to broader disease applications with other peptides where DPPIV-inhibitors are not prescribed. In conclusion, this work demonstrates the power of chemical-biotechnology in its ability to refine macromolecular structure in a manner that is not possible in nature. In this instance we demonstrate that a powerful synthetic methodology can be refined to deliver native hormone pharmacology from a physiological buffer in a manner that the natural hormone cannot. This averts the proverbial Shakespearean question, as a chemically enhanced, depsi-peptide demonstrates itself “to be, and not to be” in simultaneously delivering native clinical pharmacology with non-native pre-clinical properties.



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ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publication website at DOI: Experimental methods, analytical characterization of investigated peptides, supporting Figures S1-22 (PDF)

AUTHORS INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Notes P.A.M, J.P.M. and R.D.D are co-inventors on a patent application covering the glucagon analogues described here.

ACKNOWLEDGEMENTS The authors acknowledge the biological support of V. Gelfanov and the chemical support of J. Levy. A portion of this work was supported by research funding provided by Calibrium, LLC. R. DiMarchi is a founder of Calibrium, LLC a start-up company purchased by Novo Nordisk.

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Native Design of Soluble, Aggregation-Resistant Bioactive Peptides

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