Allosteric Domain Research Articles

EXTH-59. POTENT, SELECTIVE, AND BRAIN PENETRANT INHIBITORS OF EXTRACELLULAR DOMAIN EGFR ONCOGENIC MUTANTS EXPRESSED IN GBM DEMONSTRATE EFFICACY IN AN INTRACRANIAL PATIENT DERIVED XENOGRAFT MODEL

Abstract EGFR mutations identified in glioblastomas (GBM) occur nearly exclusively at the allosteric extracellular domain (ECD) and constitutively activate oncogenic signaling. Despite wide success in treating tumors expressing EGFR catalytic site mutants, no drug has demonstrated clinical utility against tumors expressing the extracellular domain EGFR mutants. We demonstrate that the family of ECD mutations are not only co-expressed in GBM, but that they all activate the oncogene through a similar disulfide bond-mediated receptor dimerization mechanism. This dimerization occurs independent of ligands and renders the Locked-dimer (LoDi)-EGFR insensitive to agents that target the EGFR kinase domain mutants in NSCLC. The kinase conformation induced by these ECD mutations seen in glioblastomas is both oncogenic and altered from kinase domain mutations, thus necessitating a new approach to targeting. By screening against cells expressing LoDi-EGFR mutants, we have identified the first inhibitors that potently and selectively target LoDi-EGFR mutants versus both canonical active site oncogenic mutants and wild type EGFR. Through an optimization effort, we have identified a novel family of potent and selective LoDi-EGFR mutant inhibitors that effectively penetrate the blood brain barrier (BBB) following oral dosing in preclinical studies. A leading exemplar, BDTX-GBM-001, inhibits the 5 major LoDi-EGFR mutants expressed in GBM with antiproliferative potency of ~10 nM while showing favorable selectivity versus the human kinome. When dosed orally in the intracranial GBM6 patient derived xenograft model at 50, 30, and 15 mg/kg, a dose responsive decrease in tumor growth, as well as a statistically significant increase in survival, were observed. These data support the continued evaluation of rationally designed BBB penetrant inhibitors selectively targeting the common LoDi-EGFR mutants and enable the first chance to fully test the clinical hypothesis of EGFR driver mutants in GBM.

Phospho-Mimetic Mutation at Ser602 Inactivates Human TRPA1 Channel.

The Transient Receptor Potential Ankyrin 1 (TRPA1) channel is an integrative molecular sensor for detecting environmental irritant compounds, endogenous proalgesic and inflammatory agents, pressure, and temperature. Different post-translational modifications participate in the discrimination of the essential functions of TRPA1 in its physiological environment, but the underlying structural bases are poorly understood. Here, we explored the role of the cytosolic N-terminal residue Ser602 located near a functionally important allosteric coupling domain as a potential target of phosphorylation. The phosphomimetic mutation S602D completely abrogated channel activation, whereas the phosphonull mutations S602G and S602N produced a fully functional channel. Using mutagenesis, electrophysiology, and molecular simulations, we investigated the possible structural impact of a modification (mutation or phosphorylation) of Ser602 and found that this residue represents an important regulatory site through which the intracellular signaling cascades may act to reversibly restrict or “dampen” the conformational space of the TRPA1 channel and promote its transitions to the closed state.

Kinase Domain Is a Dynamic Hub for Driving LRRK2 Allostery.

Protein kinases and GTPases are the two major molecular switches that regulate much of biology, and both of these domains are embedded within the large multi-domain Leucine-Rich Repeat Kinase 2 (LRRK2). Mutations in LRRK2 are the most common cause of familial Parkinson’s disease (PD) and are also implicated in Crohn’s disease. The recent Cryo-Electron Microscopy (Cryo-EM) structure of the four C-terminal domains [ROC COR KIN WD40 (RCKW)] of LRRK2 includes both of the catalytic domains. Although the important allosteric N-terminal domains are missing in the Cryo-EM structure this structure allows us to not only explore the conserved features of the kinase domain, which is trapped in an inactive and open conformation but also to observe the direct allosteric cross-talk between the two domains. To define the unique features of the kinase domain and to better understand the dynamic switch mechanism that allows LRRK2 to toggle between its inactive and active conformations, we have compared the LRRK2 kinase domain to Src, BRaf, and PKA. We also compare and contrast the two canonical glycine-rich loop motifs in LRRK2 that anchor the nucleotide: the G-Loop in protein kinases that anchors ATP and the P-Loop in GTPases that anchors GTP. The RCKW structure also provides a template for the cross-talk between the kinase and GTPase domains and brings new mechanistic insights into the physiological function of LRRK2 and how the kinase domain, along with key phosphorylation sites, can serve as an allosteric hub for mediating conformational changes.

Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor

The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor’s ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR’s extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.

Receptor-interacting protein kinase 1 (RIPK1) as a therapeutic target.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a key mediator of cell death and inflammation. The unique hydrophobic pocket in the allosteric regulatory domain of RIPK1 has enabled the development of highly selective small-molecule inhibitors of its kinase activity, which have demonstrated safety in preclinical models and clinical trials. Potential applications of these RIPK1 inhibitors for the treatment of monogenic and polygenic autoimmune, inflammatory, neurodegenerative, ischaemic and acute conditions, such as sepsis, are emerging. This article reviews RIPK1 biology and disease-associated mutations in RIPK1 signalling pathways, highlighting clinical trials of RIPK1 inhibitors and potential strategies to mitigate development challenges.

Surface charge of Merkel cell polyomavirus small T antigen determines cell transformation through allosteric FBW7 WD40 domain targeting

Merkel cell polyomavirus (MCV) small T (sT) is the main oncoprotein in Merkel cell carcinoma (MCC) development. A unique domain of sT, LT stabilization domain (LSD), has been reported to bind and inactivate multiple SCF (Skp1-Cullin-F-box) E3 ligases. These interactions impede the turnover of MCV large T (LT) antigen and cellular oncoproteins such as c-Myc and cyclin E, thereby promoting viral replication and cell transformation. However, it is currently unclear how this LSD region contributes to multiple transforming activities of sT. Structural docking simulation of sT and F-box and WD repeat domain-containing 7 (FBW7) revealed a novel allosteric interaction between sT and FBW7 WD40 domain. This model is supported by experimental evidence confirming that charge engineering in the LSD alters sT-WD40 binding. Specifically, loss of net positive charge in the LSD prevents sT-FBW7 binding by abrogating the electrostatic interaction, thus impeding inhibition of FBW7 by sT. Furthermore, positively charged mutations in the LSD significantly restored the sT function and its ability to transform rodent fibroblast cells. We infer that the surface charge of sT is a major determinant for targeting E3 ligases, which leads to sT-induced cell transformation, an observation that could be used to develop targeted therapeutics for MCC.

Determination of allosteric and active sites responsible for catalytic activity of delta 12 fatty acid desaturase from Geotrichum candidum and Mortierella alpina by domain swapping

Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

Molecular Dynamics Simulations of Antibiotic Ceftaroline at the Allosteric Site of Penicillin‐Binding Protein 2a (PBP2a)

AbstractMethicillin‐resistant Staphylococcus aureus (MRSA) tolerates β‐lactam antibiotics by carrying out cell wall synthesis with the transpeptidase Penicillin‐binding protein 2a (PBP2a), which cannot be inhibited by β‐lactams. It has been proposed that PBP2a's active site is protected by two loops to reduce the probability of it binding with β‐lactams. Previous crystallographic studies suggested that this protected active site opens for reaction once a native substrate binds at an allosteric domain of PBP2a. This opening was proposed for the new β‐lactam ceftaroline's mechanism in successfully treating MRSA infections, i. e. by it binding to the allosteric site, thereby opening the active site to inhibition. In this work, we investigate the binding of ceftaroline at this proposed allosteric site using molecular dynamics simulations. Unstable binding was observed using the major force fields CHARMM36 and Amber ff14SB, and free energy calculations were unable to confirm a strong allosteric effect. Our study suggests that the allosteric effect induced by ceftaroline is weak at best.

Biased allosteric modulators as a novel approach to induce biased signalling in G‐protein coupled receptors: The case of beta 2 adrenergic receptor in cardiovascular diseases

G protein‐coupled receptors (GPCRs) are the therapeutic target of nearly half of the current drugs in the market. It has been now well established that GPCRs can signal through multiple transducers, including G proteins and beta‐arrestins. These signalling pathways can be activated or blocked by “balanced” agonists or antagonists, but also, they can also be selectively activated in a “biased” response. Until now, biased signalling has been induced by biased ligands and biased receptors, any of which can result in preferential signalling through G proteins or beta‐arrestins. However, the discovery and development of GPCR biased agonists has been quite challenging, since in most GPCRs there is no structure activity relationship studies available, moreover, different active conformations for each pathway are likely very similar. Traditionally, biased agonists have been developed to target only the orthosteric site which is the binding site of the endogenous ligands. Development of biased agonists targeting the orthosteric site might be extremely difficult, especially in the absence of structure‐activity relationship information. Biased agonism holds great promise as a mechanism to significantly reduce the side effects that current drugs in used in the clinic, as well as develop drugs for use on their own. Also, ligands that produce biased signalling will serve as valuable tools for elucidation of the molecular mechanisms underlying GPCR‐signalling. Allosteric modulators are ligands which bind to a receptor at a site distinct from that of the endogenous agonist and they do not activate the receptor rather than stabilize or induce intermediate active or inactive structural conformations modulating signalling of the orthosteric ligands. We tested the hypothesis that biased signalling of the receptor could be achieved by targeting allosteric domains which show high diversity in structure and amino acid sequence among the GPCRs. The rationale is that the development of a novel series of allosteric modulators will regulate GPCR signalling in such way that the endogenous ligand would produce biased signalling only when the target receptor is bound to an allosteric modulator. In this study, we targeted the intracellular domains of the beta 2 adrenergic receptor (b2‐AR) to produce beta‐arrestin biased signalling when activated by traditional orthosteric ligands. A highly selective b2‐AR biased allosteric modulator would be highly desirable to treat a number of cardiovascular diseases. Thus, we used a combination of state of art drug discovery platforms, in silico calculations and peptidomimetics to develop a beta‐arrestin biased allosteric modulator AR1981. This allosteric modulator increased the beta‐arrestin recruitment efficacy of isoproterenol by two‐fold. Also, AR1981 was able to increase the potency of isoproterenol in beta‐arrestin recruitment by more than ten‐fold. Thus, AR1981 is a promising tool to access a novel pharmacological profile stimulating cardioprotective signalling through the b2‐AR and can serve as a model for the next generation of cardiovascular drug development. The outcome of the proposed work will facilitate the development of a new generation of allosteric modulators for GPCRs.

Design, Synthesis and Characterization of a New Series of Fluorescent Metabotropic Glutamate Receptor Type 5 Negative Allosteric Modulators.

In recent years, new drug discovery approaches based on novel pharmacological concepts have emerged. Allosteric modulators, for example, target receptors at sites other than the orthosteric binding sites and can modulate agonist-mediated activation. Interestingly, allosteric regulation may allow a fine-tuned regulation of unbalanced neurotransmitter’ systems, thus providing safe and effective treatments for a number of central nervous system diseases. The metabotropic glutamate type 5 receptor (mGlu5R) has been shown to possess a druggable allosteric binding domain. Accordingly, novel allosteric ligands are being explored in order to finely regulate glutamate neurotransmission, especially in the brain. However, before testing the activity of these new ligands in the clinic or even in animal disease models, it is common to characterize their ability to bind mGlu5Rs in vitro. Here, we have developed a new series of fluorescent ligands that, when used in a new NanoBRET-based binding assay, will facilitate screening for novel mGlu5R allosteric modulators.

A regulatory SH2 domain-targeting protein binder effectively inhibits the activity of Bruton’s tyrosine kinase and its drug-resistant variants

Human Bruton’s tyrosine kinase (hBtk) plays a key role in growth and metabolism of B cells, but its dysfunctions cause various B-cell malignancies. Inhibitors targeting the ATP-binding pocket of hBtk have been developed, but they have several drawbacks such as adverse side effects and occurrence of drug-resistant mutations. Here, we present a protein binder which specifically binds to an allosteric regulatory SH2 domain of hBtk. The protein binder effectively inhibited the hBtk activity, indicating a critical role of the SH2 domain in allosteric regulation of the hBtk activity. Cytosolic delivery of the protein binder led to a significant inhibition on the BCR-mediated signaling and viability of B lymphoma cells. The utility of our approach was demonstrated by effective inhibition of drug-resistant hBtk variants by the protein binder. Based on the computationally predicted binding mode, the protein binder is likely to inhibit the hBtk activity by disrupting the interaction between the SH2 domain and kinase domain. The present approach can be used for developing therapeutic agents with improved efficacy for B-cell lymphoma.

Evidence for an Allosteric S-Nitrosoglutathione Binding Site in S-Nitrosoglutathione Reductase (GSNOR).

Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill–Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn2+-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen–deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme.

Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2-E1 dissociation during viral entry.

Receptor binding is the first step in viral cell entry. In enveloped virus cell entry, viral and host membrane fusion follows receptor binding. Viral surface receptor-binding protein associates with membrane fusion protein and masks its structure, to prevent pre-mature fusion activity. Dissociation of receptor-binding protein from fusion protein is an essential step before membrane fusion. Mechanism of receptor binding leading to dissociation of receptor binding and fusion protein is poorly understood in alphaviruses. Chikungunya virus (CHIKV), an alphavirus, re-emerged as a global pathogen in recent past. CHIKV surface envelope proteins, E2 and E1, function as receptor binding and fusion protein, respectively. Site of heparan sulfate (HS) receptor binding on E2–E1 heterodimer and its effect on E2–E1 heterodimer conformation is not known. Using molecular docking, we mapped HS binding to a positively charged pocket on E2 that is structurally conserved in alphaviruses. Based on our results from docking and sequence analysis, we identified a novel HS-binding sequence motif in E2. Purified E2 binds to heparin and HS specifically through charge interactions. Binding affinity of E2 to HS is comparable with other known HS–protein interactions (K d ∼ 1.8 μM). Mutation of charged residues in the predicted HS-binding motif of E2 to alanine resulted in reduction of HS binding. Molecular dynamics (MD) simulations on E2, after docking HS, predicted allosteric domain movements. Fluorescence spectroscopy, far-UV circular dichroism spectroscopy, fluorescence resonance energy transfer experiments on HS-bound E2 corroborate our findings from MD simulations. We propose a mechanism where receptor-binding results in allosteric domain movements in E2, explaining E2–E1 dissociation.

Cover Feature: Computational Design and Study of Artificial Selenoenzyme with Controllable Activity Based on an Allosteric Protein Scaffold (Chem. Eur. J. 44/2019)

A computational protocol that combines docking, molecular dynamics, and MM-PBSA was described to guide the design of an artificial selenoenzyme based on a Ca2+-responsive recoverin (Rn) protein. The catalytically active site of glutathione peroxidase (GPx) can be precisely incorporated into the allosteric domain of Rn, leading to a high GPx activity and tightly controlled function for the catalyzed reduction of H2O2 by glutathione (GSH). These results provide important insights into how to create an enzyme-like site in non-homologous protein scaffolds. More information can be found in the Full Paper by Q. Luo et al. on page 10350.

Computational Design and Study of Artificial Selenoenzyme with Controllable Activity Based on an Allosteric Protein Scaffold.

The establishment of new enzymatic function in an existing scaffold is a great challenge for protein engineers. In previous work, a highly efficient artificial selenoenzyme with controllable activity was constructed, based on a Ca2+ -responsive recoverin (Rn) protein. In this study, a design strategy combining docking, molecular dynamics, and MM-PBSA is presented, to predict the catalytically active site of glutathione peroxidase (GPx) on the allosteric domain of Rn. The energy contributions of the binding hot spot residues are evaluated further by energy decomposition analysis to determine the detailed substrate recognition mechanism of Rn, which provides clear guidance for artificial enzyme design for improved substrate binding (Michaelis-Menten constant, Km ).

Allosteric autoactivation of SOS and its kinetic mechanism

ABSTRACT Son of Sevenless (SOS), one of guanine nucleotide exchange factors (GEFs), activates Ras. We discovered that the allosteric domain of SOS yields SOS to proceed a previously unrecognized autoactivation kinetics. Its essential feature is a time-dependent acceleration of SOS feedback activation with a reaction initiator or with the priming of active Ras. Thus, this mechanistic autoactivation feature explains the notion, previously only conjectured, of accelerative SOS activation followed by the priming of active Ras, an action produced by another GEF Ras guanyl nucleotide-releasing protein (RasGRP). Intriguingly, the kinetic transition from gradual RasGRP activation to accelerative SOS activation has been interpreted as an analog to digital conversion; however, from the perspective of autoactivation kinetics, it is a process of straightforward RasGRP-mediated SOS autoactivation. From the viewpoint of allosteric protein cooperativity, SOS autoactivation is a unique time-dependent cooperative SOS activation because it enables an active SOS to accelerate activation of other SOS as a function of time. This time-dependent SOS cooperativity does not belong to the classic steady-state protein cooperativity, which depends on ligand concentration. Although its hysteretic or sigmoid-like saturation curvature is a classic hallmark of steady-state protein cooperativity, its hyperbolic saturation figure typically represents protein noncooperativity. We also discovered that SOS autoactivation perturbs the previously predicted hysteresis of SOS activation in a steady state to produce a hyperbolic saturation curve. We interpret this as showing that SOS allostery elicits, through SOS autoactivation, cooperativity uniquely time-dependent but not ligand concentration dependent.

Characterization of Temperature-Dependent Gating in Archaebacterial Calcium Activated Potassium Channel

MthK is an archaebacterial K+ channel, which is gated by large cytoplasmic RCK domains that facilitate Ca2+-activation of the channel. This bacterial channel is a good model for studying the mechanisms underlying calcium dependent gating in ion channels because it tractable to both structural and functional studies. An earlier study has also suggested that the calcium-dependent gating of MthK is also regulated by temperature (Parfenova, L. V. et al. (2006). J Biol Chem 281, 21131-21138). Here, using inside-out patch-clamping recording of inactivation removed MthK from spheroplasts, I have characterized the temperature-dependence of MthK channel at varying Ca2+ concentrations. Our preliminary data shows that the activation of this inactivation-removed channel is highly temperature-dependent. We have also purified and reconstituted this inactivation removed MthK in soybean polar lipids and found similar temperature-dependence albeit at lower concentrations of calcium. This suggests that the temperature-dependence of MthK is intrinsic. Furthermore, allosteric analysis of electrophysiological data shows that temperature unexpectedly modulates coupling between Ca2+ sensor and the pore rather than acting through a completely independent allosteric domain.

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion.

All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an “off” to an “on” state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.

The Molecular Organization of Human cGMP Specific Phosphodiesterase 6 (PDE6): Structural Implications of Somatic Mutations in Cancer and Retinitis Pigmentosa

In the cyclic guanosine monophosphate (cGMP) signaling pathway, phosphodiesterase 6 (PDE6) maintains a critical balance of the intracellular concentration of cGMP by catalyzing it to 5′ guanosine monophosphate (5′-GMP). To gain insight into the mechanistic impacts of the PDE6 somatic mutations that are implicated in cancer and retinitis pigmentosa, we first defined the structure and organization of the human PDE6 heterodimer using computational comparative modelling. Each subunit of PDE6αβ possesses three domains connected through long α-helices. The heterodimer model indicates that the two chains are likely related by a pseudo two-fold axis. The N-terminal region of each subunit is comprised of two allosteric cGMP-binding domains (Gaf-A & Gaf-B), oriented in the same way and interacting with the catalytic domain present at the C-terminal in a way that would allow the allosteric cGMP-binding domains to influence catalytic activity. Subsequently, we applied an integrated knowledge-driven in silico mutation analysis approach to understand the structural and functional implications of experimentally identified mutations that cause various cancers and retinitis pigmentosa, as well as computational saturation mutagenesis of the dimer interface and cGMP-binding residues of both Gaf-A, and the catalytic domains. We studied the impact of mutations on the stability of PDE6αβ structure, subunit-interfaces and Gaf-cGMP interactions. Further, we discussed the changes in interatomic interactions of mutations that are destabilizing in Gaf-A (R93L, V141 M, F162 L), catalytic domain (D600N, F742 L, F776 L) and at the dimer interface (F426A, F248G, F424 N). This study establishes a possible link of change in PDE6αβ structural stability to the experimentally observed disease phenotypes.

GPCR drug discovery-moving beyond the orthosteric to the allosteric domain.

Allosteric modulation of G protein coupled receptors (GPCRs) is rapidly becoming a standard option for development of therapeutics headed to the clinic. Although GPCRs represent about 35% of marketed drugs, to date only two allosteric modulators have been approved for human use. However, many are now in early clinical development are can provide unique regulation of GPCRs including high selectivity along with physiologic temporal and spatial signaling. These molecules bind to a site that is distinct from the site where the endogenous agonist binds yet can provide robust modulation effects that span from the positive to the negative. Along with classical chemogenomic techniques, newer technology is being directly applied to their development including three dimensional biophysical structure-function analysis and in silico molecular dynamic simulations. The goal is to provide rationally designed molecules from well informed physical and in silico data to speed the discovery and development of the next generation therapeutics. In this chapter an example of the evolution of allosteric drug discovery targeting the muscarinic receptor family should serve to inform of progress in this exciting area of research and early drug development.