Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L-11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a ß site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the ß site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).

Analysis of neutral mutational drift in an allosteric enzyme.

Neutral mutational drift is an important source of biological diversity that remains underexploited in fundamental studies of protein biophysics. This study uses a synthetic transcriptional circuit to study neutral drift in protein tyrosine phosphatase 1B (PTP1B), a mammalian signaling enzyme for which conformational changes are rate limiting. Kinetic assays of purified mutants indicate that catalytic activity, rather than thermodynamic stability, guides enrichment under neutral drift, where neutral or mildly activating mutations can mitigate the effects of deleterious ones. In general, mutants show a moderate activity-stability tradeoff, an indication that minor improvements in the activity of PTP1B do not require concomitant losses in its stability. Multiplexed sequencing of large mutant pools suggests that substitutions at allosterically influential sites are purged under biological selection, which enriches for mutations located outside of the active site. Findings indicate that the positional dependence of neutral mutations within drifting populations can reveal the presence of allosteric networks and illustrate an approach for using synthetic transcriptional systems to explore these mutations in regulatory enzymes.

A biophysical rationale for the selective inhibition of PTP1B over TCPTP by nonpolar terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including type 2 diabetes, obesity, and cancer. However, a high degree of structural similarity between the catalytic domains of these enzymes has made the development of selective pharmacological inhibitors an enormous challenge. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over TCPTP, two PTPs with high sequence conservation. Here, we use molecular modeling with experimental validation to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations indicate that PTP1B and TCPTP contain a conserved h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically influential WPD loop, which it links to the L-11 loop and α 3 and α 7 helices-the C-terminal side of the catalytic domain. Terpenoid binding to either of two proximal allosteric sites-an α site and a ß site-can disrupt the allosteric network. Interestingly, binding to the α site forms a stable complex with only PTP1B; in TCPTP, where two charged residues disfavor binding at the α site, the terpenoids bind to the ß site, which is conserved between the two proteins. Our findings indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate, more broadly, how minor differences in the conservation of neighboring-yet functionally similar-allosteric sites can have very different implications for inhibitor selectivity.

Allosteric Inhibition of PTP1B by a Nonpolar Terpenoid.

Protein tyrosine phosphatases (PTPs) are promising drug targets for treating a wide range of diseases such as diabetes, cancer, and neurological disorders, but their conserved active sites have complicated the design of selective therapeutics. This study examines the allosteric inhibition of PTP1B by amorphadiene (AD), a terpenoid hydrocarbon that is an unusually selective inhibitor. Molecular dynamics (MD) simulations carried out in this study suggest that AD can stably sample multiple neighboring sites on the allosterically influential C-terminus of the catalytic domain. Binding to these sites requires a disordered α7 helix, which stabilizes the PTP1B-AD complex and may contribute to the selectivity of AD for PTP1B over TCPTP. Intriguingly, the binding mode of AD differs from that of the most well-studied allosteric inhibitor of PTP1B. Indeed, biophysical measurements and MD simulations indicate that the two molecules can bind simultaneously. Upon binding, both inhibitors destabilize the α7 helix by disrupting interactions at the α3-α7 interface and prevent the formation of hydrogen bonds that facilitate closure of the catalytically essential WPD loop. These findings indicate that AD is a promising scaffold for building allosteric inhibitors of PTP1B and illustrate, more broadly, how unfunctionalized terpenoids can engage in specific interactions with protein surfaces.

Analysis of Three Architectures for Controlling PTP1B with Light.

Photosensory domains are powerful tools for placing proteins under optical control, but their integration into light-sensitive chimeras is often challenging. Many designs require structural iterations, and direct comparisons of alternative approaches are rare. This study uses protein tyrosine phosphatase 1B (PTP1B), an influential regulatory enzyme, to compare three architectures for controlling PTPs with light: a protein fusion, an insertion chimera, and a split construct. All three designs permitted optical control of PTP1B activity in vitro (i.e., kinetic assays of purified enzyme) and in mammalian cells; photoresponses measured under both conditions, while different in magnitude, were linearly correlated. The fusion- and insertion-based architectures exhibited the highest dynamic range and maintained native localization patterns in mammalian cells. A single insertion architecture enabled optical control of both PTP1B and TCPTP, but not SHP2, where the analogous chimera was active but not photoswitchable. Findings suggest that PTPs are highly tolerant of domain insertions and support the use of in vitro screens to evaluate different optogenetic designs.

Optogenetic interrogation and control of cell signaling.

Signaling networks control the flow of information through biological systems and coordinate the chemical processes that constitute cellular life. Optogenetic actuators - genetically encoded proteins that undergo light-induced changes in activity or conformation - are useful tools for probing signaling networks over time and space. They have permitted detailed dissections of cellular proliferation, differentiation, motility, and death, and enabled the assembly of synthetic systems with applications in areas as diverse as photography, chemical synthesis, and medicine. In this review, we provide a brief introduction to optogenetic systems and describe their application to molecular-level analyses of cell signaling. Our discussion highlights important research achievements and speculates on future opportunities to exploit optogenetic systems in the study and assembly of complex biochemical networks.

In-Situ Molecular Vapor Composition Measurements During Lyophilization.

PURPOSE: Monitoring process conditions during lyophilization is essential to ensuring product quality for lyophilized pharmaceutical products. Residual gas analysis has been applied previously in lyophilization applications for leak detection, determination of endpoint in primary and secondary drying, monitoring sterilization processes, and measuring complex solvents. The purpose of this study is to investigate the temporal evolution of the process gas for various formulations during lyophilization to better understand the relative extraction rates of various molecular compounds over the course of primary drying. METHODS: In this study, residual gas analysis is used to monitor molecular composition of gases in the product chamber during lyophilization of aqueous formulations typical for pharmaceuticals. Residual gas analysis is also used in the determination of the primary drying endpoint and compared to the results obtained using the comparative pressure measurement technique. RESULTS: The dynamics of solvent vapors, those species dissolved therein, and the ballast gas (the gas supplied to maintain a set-point pressure in the product chamber) are observed throughout the course of lyophilization. In addition to water vapor and nitrogen, the two most abundant gases for all considered aqueous formulations are oxygen and carbon dioxide. In particular, it is observed that the relative concentrations of carbon dioxide and oxygen vary depending on the formulation, an observation which stems from the varying solubility of these species. This result has implications on product shelf life and stability during the lyophilization process. CONCLUSIONS: Chamber process gas composition during lyophilization is quantified for several representative formulations using residual gas analysis. The advantages of the technique lie in its ability to measure the relative concentration of various species during the lyophilization process. This feature gives residual gas analysis utility in a host of applications from endpoint determination to quality assurance. In contrast to other methods, residual gas analysis is able to determine oxygen and water vapor content in the process gas. These compounds have been shown to directly influence product shelf life. With these results, residual gas analysis technique presents a potential new method for real-time lyophilization process control and improved understanding of formulation and processing effects for lyophilized pharmaceutical products.