Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications.

Toxic misfolding of proinsulin variants in ß-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking ß-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.

Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation.

The mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin's biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of ß-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin's first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin's putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.

Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding.

Domain-minimized insulin receptors (IRs) have enabled crystallographic analysis of insulin-bound "micro-receptors." In such structures, the C-terminal segment of the insulin B chain inserts between conserved IR domains, unmasking an invariant receptor-binding surface that spans both insulin A and B chains. This "open" conformation not only rationalizes the inactivity of single-chain insulin (SCI) analogs (in which the A and B chains are directly linked), but also suggests that connecting (C) domains of sufficient length will bind the IR. Here, we report the high-resolution solution structure and dynamics of such an active SCI. The hormone's closed-to-open transition is foreshadowed by segmental flexibility in the native state as probed by heteronuclear NMR spectroscopy and multiple conformer simulations of crystallographic protomers as described in the companion article. We propose a model of the SCI's IR-bound state based on molecular-dynamics simulations of a micro-receptor complex. In this model, a loop defined by the SCI's B and C domains encircles the C-terminal segment of the IR α-subunit. This binding mode predicts a conformational transition between an ultra-stable closed state (in the free hormone) and an active open state (on receptor binding). Optimization of this switch within an ultra-stable SCI promises to circumvent insulin's complex global cold chain. The analog's biphasic activity, which serendipitously resembles current premixed formulations of soluble insulin and microcrystalline suspension, may be of particular utility in the developing world.

An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein.

Thermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.

An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models.

While it is increasingly recognized that three-dimensional (3D) cell culture models recapitulate drug responses of human cancers with more fidelity than monolayer cultures, a lack of quantitative analysis methods limit their implementation for reliable and routine assessment of emerging therapies. Here, we introduce an approach based on computational analysis of fluorescence image data to provide high-content readouts of dose-dependent cytotoxicity, growth inhibition, treatment-induced architectural changes and size-dependent response in 3D tumour models. We demonstrate this approach in adherent 3D ovarian and pancreatic multiwell extracellular matrix tumour overlays subjected to a panel of clinically relevant cytotoxic modalities and appropriately designed controls for reliable quantification of fluorescence signal. This streamlined methodology reads out the high density of information embedded in 3D culture systems, while maintaining a level of speed and efficiency traditionally achieved with global colorimetric reporters in order to facilitate broader implementation of 3D tumour models in therapeutic screening.

PDT dose parameters impact tumoricidal durability and cell death pathways in a 3D ovarian cancer model.

The successful implementation of photodynamic therapy (PDT)-based regimens depends on an improved understanding of the dosimetric and biological factors that govern therapeutic variability. Here, the kinetics of tumor destruction and regrowth are characterized by systematically varying benzoporphyrin derivative (BPD)-light combinations to achieve fixed PDT doses (M × J cm(-2)). Three endpoints were used to evaluate treatment response: (1) Viability evaluated every 24 h for 5 days post-PDT; (2) Photobleaching assessed immediately post-PDT; and (3) Caspase-3 activation determined 24 h post-PDT. The specific BPD-light parameters used to construct a given PDT dose significantly impact not only acute cytotoxic efficacy, but also treatment durability. For each dose, PDT with 0.25 µM BPD produces the most significant and sustained reduction in normalized viability compared to 1 and 10 µM BPD. Percent photobleaching correlates with normalized viability for a range of PDT doses achieved within BPD concentrations. To produce a cytotoxic response with 10 µM BPD that is comparable to 0.25 and 1 µM BPD a reduction in irradiance from 150 to 0.5 mW cm(-2) is required. Activated caspase-3 does not correlate with normalized viability. The parameter-dependent durability of outcomes within fixed PDT doses provides opportunities for treatment customization and improved therapeutic planning.

Image-Based Quantification of Benzoporphyrin Derivative Uptake, Localization, and Photobleaching in 3D Tumor Models, for Optimization of PDT Parameters.

Photodynamic therapy (PDT) is a light-based treatment modality in which wavelength specific activation of a photosensitizer (PS) generates cytotoxic response in the irradiated region. PDT response is critically dependent on several parameters including light dose, PS dose, uptake time, fluence rate, and the mode of light delivery. While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response. Here we present a theranostic technique, combining the inherent ability of the PS to serve simultaneously as a therapeutic and imaging agent, with the use of image-based treatment assessment in three dimensional (3D) in vitro tumor models, to comprise a platform to evaluate the impact of PDT parameters on treatment outcomes. We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells. Informed by photobleaching patterns and correlation with cytotoxic response, asymmetric fractionated light delivery at 4 hours BPD uptake was found to be the most effective regimen assessed. Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules. The framework introduced here provides a means for systematic assessment of PDT treatment parameters in biologically relevant 3D tumor models with potential for broader application to other systems.