Microcanonical insights into the physicochemical stability of the coformulation of insulin with amylin analogues.

Injections of insulin are the main treatment for diabetes, but in the long run this therapy can induce serious drawbacks. This has inspired new drugs able to decrease insulin requirements. For instance, human amylin (hIAPP) is a small hormone cosecreted by pancreatic ß-cells with insulin to which is a synergistic partner. However, the high amyloidogenicity of hIAPP precluded it as a therapeutics and led to the design of pramlintide (sIAPP), a chimeric analogue with substitutions (A25P, S28P, and S29P) inherited from the aggregation-resistant rat isoform (rIAPP). Despite sIAPP advantages, it still shares with hIAPP a poorly soluble profile at physiological pH that hampers its mixture with insulin. Recent improvements, as charge-enhanced mutants, have been proposed. For instance, sIAPP+ was screened in silico by purely microcanonical thermostatistical methods and adds to sIAPP an S20R mutation to uplift its solubility. This suggests that such physically inspired computational approach may also be auspicious on devising effective coformulations of insulin with amylin analogues. In this seminal attempt, we make comparative multicanonical simulations of regular acting human insulin coformulated with hIAPP, sIAPP, or sIAPP+. To assess the respective physicochemical stabilities against aggregation, we characterize the structural-phase transitions through the microcanonical thermodynamic formalism and evaluate their time lags using the classical nucleation theory. These results are then correlated with estimates of solvation free energies, modeled by the Poisson-Boltzmann equation, and structural propensities. Experimental essays are compared to our simulations and support our methodology.

Occurrence of Biased Conformations as Precursors of Assembly States in Fibril Elongation of Amyloid-ß Fibril Variants: An In Silico Study.

We investigate the prevalence, and so the role in the amyloidogenesis, of biased conformations in large ensembles of monomeric forms for Aß42 and Aß40 that can trigger the formation and growth of fibrils described by a dock-lock mechanism. We model such biased conformations as the structural monomeric units that constitute the Protein Data Bank fibrils 2beg, 2mxu, and 2lmn. These units were employed as templates to search for similar structures in statistical conformational ensembles of Aß peptides generated by molecular dynamics with an accurate force field in explicit solvation, whose high quality is revealed by comparison with residual dipolar coupling (RDC) experiments. The conformational ensembles generated by these intrinsically disordered peptides do not contain conformations highly similar to the amyloidogenic templates. This is a consequence of the low thermodynamic stability exhibited by the template-like conformations. A further constant-pH Monte Carlo study has revealed that this stability can be increased by suitable pH conditions, which helps to trigger the fibril elongation. Moreover, our analyses on the free energy landscapes, hydrogen bond prevalences, and principal component analysis distributions emphasize the relevance of many-body long-range cooperative interactions, likely acting over the infrequent preexisting structurally biased conformations, to explain the fibrils' emergence.

Synergistic long-range effects of mutations underlie aggregation propensities of amylin analogues.

The USFDA has approved pramlintide, commercially named Symlin (sIAPP), as adjunctive therapy for type 2 diabetes (T2D). This analogue of the human amylin peptide (hIAPP) has triple proline substitutions typical of the rat isoform (rIAPP). Recently, it was proposed that pramlintide solubility and aggregation resistance might be improved by incorporating further mutations, as S20R, screened from the wild-type porcine isoform (pIAPP), which leads to the variant named sIAPP+. To better elucidate how such properties might be systematically induced in rationally designed analogues, we performed comparative assessments of rIAPP, sIAPP, and sIAPP+ using replica-exchange molecular dynamics (REMD) with an accurate combination of force field Charmm22* and explicit aqueous solvation TIP4P/Ew. Our thermo-structural analyses show that sIAPP exhibits a thermal conversion channel of helices[Formula: see text]-sheets resembling hIAPP. This channel is depleted in rIAPP and is absent in sIAPP+. As a consequence, sIAPP+ presents an overall decrease of ß-like secondary structures and an overstabilization of α-helices. Additionally, we observed in rIAPP and sIAPP+ an increase in the backbone RMSF of molecular terminals and the exposed area of key residues. These structural features of sIAPP+ suggest a nonamyloidogenic character, which is corroborated by our judicious estimate of the electrostatic component of the solvation free energy using a generalized Born model, and so it may constitute an alternative strategy to sIAPP as a peptide analogue of hIAPP. Furthermore, our findings confirm that different aggregation propensities of amylin and its analogues are synergistically modulated by long-range effects of key mutations. Graphical Abstract S20R-Pramlintide.

In Silico Comparative Study of Human and Porcine Amylin.

Islet transplantation is a promising treatment for type 2 diabetes, but its success is impaired by progressive graft loss, likely due to cytotoxic aggregation of the hormone human islet amyloid polypeptide (IAPP) secreted by the endocrine pancreas. Alternatively, the effectiveness of porcine xenotransplantations might be explained by the fibrillization-resistance of the porcine mutant. To better elucidate such molecular mechanisms, we performed comparative replica-exchange molecular dynamics simulations of both human (hIAPP) and porcine (pIAPP) isoforms. The accurate force field Charmm22* with explicit aqueous solvation TIP4P/Ew ensured a minimal structural bias around physiological temperatures. Along which, the peptides are shown to present no structural-phase transition of folding from a microcanonical thermodynamics perspective. Both IAPP isoforms predominantly exhibit random-coil structures, but in a minor percentage we observed a direct α-helix → ß-sheet thermal conversion during the folding process of hIAPP, which is absent in pIAPP. The amyloidogenic segment 20-29 in pIAPP, which hosts 5 out of the 10 overall mutations found in this peptide, is strongly depleted of ß-sheet structures in constrast to hIAPP. Hydrogen bond analysis revealed a predominant frequency of 3-helix contacts in this residue range for pIAPP. These features of pIAPP anticorrelate with the presence of a well-known ß-sheet rich monomeric state that in hIAPP acts as an intermediate inducing oligomerization.

Structural Interconversion in Alzheimer's Amyloid-ß(16-35) Peptide in an Aqueous Solution.

Structural properties of Aß(16-35) fragment are investigated as a model for the amyloid-ß peptide excluding its coil-inducing terminals. Our replica-exchange molecular dynamics simulations using all-atom and explicit aqueous solvation widely reduce any structural bias. The principal folding pathway shows direct conversion of coil to ß-sheet, without the long proposed helix intermediates. Our principal component analysis indicates that the fragment is also intrinsically disordered, as the full amyloid-ß peptide. Thus, the observed folding mechanism lacks free-energy barriers and any peaks in the thermal capacity.

Be positive: optimizing pramlintide from microcanonical analysis of amylin isoforms.

Amylin, or human islet amyloid polypeptide (hIAPP), is a 37-residue hormone synergistic to insulin and co-secreted with it by ß-cells in the pancreas. The deposition of its cytotoxic amyloid fibrils is strongly related to the progression of Type II diabetes (T2D) and islet graft failures. Notably, isoforms from some mammalian species, such as rats (rIAPP) and porcine (pIAPP), present a few key mutations preventing aggregation. This has lead to biotechnological development of drugs for adjunct therapies of T2D, such as pramlintide, a variant of hIAPP inspired by rIAPP whose proline substitutions have ß-strand fibril-breaking properties. Ideally, such a drug should be formulated with insulin and co-administered, but this has been prevented by a poor solubility profile at the appropriate pH. Hopefully, this could be improved with appropriate point mutations, increasing the molecular net charge. Despite experimental progress, preliminary screening during rational drug design can greatly benefit from thermodynamic insight derived from molecular simulations. So we introduce microcanonical thermostatistics analysis of multicanonical (MUCA) simulations of wild-type amylin isoforms as a systematic assessment of protein thermostability. As a consequence of this comprehensive investigation, the most suitable single-point mutations able to optimize pramlintide are located among the wild-type amylin isoforms. In particular, we find that aggregation inhibition and increased solubility are inherited by pramlintide through further S20R substitution typical of pIAPP. Thus, we provide a consistent thermostatistical methodology to aid the design of improved adjunct therapies for T2D according to current clinical knowledge.

Breakout character of islet amyloid polypeptide hydrophobic mutations at the onset of type-2 diabetes.

Toxic fibrillar aggregates of islet amyloid polypeptide (IAPP) appear as the physical outcome of a peptidic phase transition signaling the onset of type-2 diabetes mellitus in different mammalian species. In particular, experimentally verified mutations on the amyloidogenic segment 20-29 in humans, cats, and rats are highly correlated with the molecular aggregation propensities. Through a microcanonical analysis of the aggregation of IAPP_{20-29} isoforms, we show that a minimalist one-bead hydrophobic-polar continuum model for protein interactions properly quantifies those propensities from free-energy barriers. Our results highlight the central role of sequence-dependent hydrophobic mutations on hot spots for stabilization, and thus for the engineering, of such biological peptides.

Microcanonical thermostatistics of coarse-grained proteins with amyloidogenic propensity.

The formation of fibrillar aggregates seems to be a common characteristic of polypeptide chains, although the observation of these aggregates may depend on appropriate experimental conditions. Partially folded intermediates seem to have an important role in the generation of protein aggregates, and a mechanism for this fibril formation considers that these intermediates also correspond to metastable states with respect to the fibrillar ones. Here, using a coarse-grained (CG) off-lattice model, we carry out a comparative analysis of the thermodynamic aspects characterizing the folding transition with respect to the propensity for aggregation of four different systems: two isoforms of the amyloid ß-protein, the Src SH3 domain, and the human prion proteins (hPrP). Microcanonical analysis of the data obtained from replica exchange method is conducted to evaluate the free-energy barrier and latent heat in these models. The simulations of the amyloid ß isoforms and Src SH3 domain indicated that the folding process described by this CG model is related to a negative specific heat, a phenomenon that can only be verified in the microcanonical ensemble in first-order phase transitions. The CG simulation of the hPrP heteropolymer yielded a continuous folding transition. The absence of a free-energy barrier and latent heat favors the presence of partially unfolded conformations, and in this context, this thermodynamic aspect could explain the reason why the hPrP heteropolymer is more aggregation-prone than the other heteropolymers considered in this study. We introduced the hydrophobic radius of gyration as an order parameter and found that it can be used to obtain reliable information about the hydrophobic packing and the transition temperatures in the folding process.