- Email: [email protected]
Substrate-Selective Enzyme Inhibitors
Trends in Pharmacological Sciences
resolve a long-standing discrepancy between human and mouse immunology, where IL-13 had been known to drive IgE switching in human B cells but not in mouse B cells. Further supporting a role for IL-13, Gowthaman et al. demonstrated that a subset of activated mouse B cells in the germinal center expressed the IL-13 receptor and could be induced to switch to IgE in the presence of IL-13. Gowthaman et al. also observed that high-affinity IgE was significantly higher in T-DOCK8-/- mice, while high-affinity IgG1 was lower compared to that in the control group. As a recent study has shown that high-affinity-IgE-producing plasma cells can differentiate from IgG1+ memory B cells upon reexposure to allergens [9], the loss of high-affinity IgG1 in T-DOCK8-/- mice indicates that Tfh13 cells induce a major population of IgG1+ B cells switching to IgE-secreting B cells. A key question is whether IL-13 produced by Tfh13 cells helps IgG1+ memory B cells switch to IgE-secreting plasma cells. However, whether the switch to IgE occurs from high-affinity IgG1 memory cells was not addressed. If IgG1+ memory B cells display enriched expression of IL-13 receptor, it could provide a mechanism for this selective response. Intriguingly, Gowthaman et al. showed that helminth infections could not induce Tfh13 cells or high-affinity IgE, although strong Th2 responses developed. Thus, Th2 cell responses do not always correlate with Tfh13 cell responses. What are the mechanisms for why helminth infections are different from allergies in terms of induction of Tfh13 cells? Solving this question can help us better understand allergies and may also help to develop specific therapeutics for different Th2-mediated diseases such as asthma and atopic dermatitis.
716
Currently, the only treatment that can prevent the mast cell degranulation response that leads to an anaphylactic shock is nonspecific IgE depletion therapy using omalizumab, a monoclonal antibody that inhibits IgE from binding to mast cells [10]. Treatments that could specifically inhibit the generation of high-affinity IgE could provide an alternative therapeutic strategy to block severe allergic responses and anaphylaxis. Since Tfh13 cells express high levels of GATA3, the use of a GATA3 DNAzyme treatment to downregulate Tfh13 cells and high-affinity IgE may have therapeutic benefit for severe allergies. Gowthaman et al. note that clinical trials of a GATA3 DNAzyme have efficacy in reducing eosinophilia, but it is unknown if this drug could affect Tfh13 or IgE responses. Ultimately, understanding the cellular mechanisms for how B cells are stimulated to produce high-affinity IgE in atopic diseases is a clinically important area of immunology research. Eisenbarth and colleagues have now made a major breakthrough on this important question, revealing how a new T cell population, Tfh13 cells, is critical for high-affinity IgE development. These findings lay the groundwork for a deeper understanding of the regulation of high-affinity IgE and anaphylaxis, and provide the basis for precision immunotherapy of atopic diseases. 1Department
of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA *Correspondence: [email protected] https://doi.org/10.1016/j.tips.2019.08.007 ª 2019 Published by Elsevier Ltd.
2. Asher, M.I. et al. (2006) Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry crosssectional surveys. Lancet 368, 733–743 3. Iweala, O.I. and Burks, A.W. (2016) Food allergy: our evolving understanding of its pathogenesis, prevention, and treatment. Curr. Allergy Asthma Rep. 16, 37 4. Blank, U. and Rivera, J. (2006) Assays for regulated exocytosis of mast cell granules. Curr. Protoc. Cell Biol. 15, 11 5. Kobayashi, T. et al. (2017) Follicular helper T cells mediate IgE antibody response to airborne allergens. J. Allergy Clin. Immunol. 139, 300–313 6. Xiong, H. et al. (2012) Sequential class switching is required for the generation of high affinity IgE antibodies. J. Exp. Med. 209, 353–364 7. Gowthaman, U. et al. (2019) Identification of a T follicular helper cell subset that drives anaphylactic IgE. Science. Published online August 1, 2019. https://doi.org/10.1126/ science.aaw6433 8. Vinuesa, C.G. et al. (2016) Follicular helper T cells. Annu. Rev. Immunol. 34, 335–368 9. He, J.S. et al. (2017) IgG1 memory B cells keep the memory of IgE responses. Nat. Commun. 8, 641 10. Kawakami, T. and Blank, U. (2016) From IgE to omalizumab. J. Immunol. 197, 4187–4192
Spotlight
Substrate-Selective Enzyme Inhibitors Qian Chu,1 Tina Chang,1 and Alan Saghatelian1,* Enzymes with multiple substrates pose a unique challenge for drug development because of an increased potential for on-target side effects. Maianti and colleagues (Nat. Chem. Biol., 2019) identify novel exo-site inhibitors with abilities to alter the substrate-selectivity of insulindegrading enzymes (IDE). Their work illuminates new therapeutic avenues for discovering small-molecule enzyme inhibitors and redefines our current understanding of drugging enzymes with multiple substrates.
References 1. Sicherer, S.H. and Sampson, H.A. (2007) Peanut allergy: emerging concepts and approaches for an apparent epidemic. J. Allergy Clin. Immunol. 120, 491–503
Trends in Pharmacological Sciences, October 2019, Vol. 40, No. 10
Biochemistry textbooks often depict biochemical pathways as a one-to-one relationship between an enzyme and a
Trends in Pharmacological Sciences
Figure 1. Comparison of Exo-Site Inhibitors to Active-Site Inhibitors. Many enzymes contain exo sites, small-molecule binding sites that do not overlap with the active site, that serve as a binding site for substrates. Activesite inhibitors (orange, right side) block all substrate turnover and, in the case of enzymes with multiple substrates (black, red substrates), this can lead to on-target side effects (inhibition of the red substrate). By contrast, exo-site inhibitors (blue, left) can reprogram an enzyme to become substrate selective, inhibiting the processing of the beneficial black substrate while allowing enzymatic processing of the deleterious red substrate, to mitigate side effects. By demonstrating the feasibility of developing substrate-selective exo-site inhibitors, Maianti and colleagues introduce a new concept into the drug discovery toolbox.
substrate [1]. While this may be true for enzymes involved in small molecule metabolism, protein-modifying enzymes (e.g., proteases, ubiquitin ligases, and kinases) almost always have multiple substrates [2–4]. In these systems, biological phenotypes may arise from the coordinated regulation of multiple substrates, with different substrates contributing to distinct cellular or physiological pathways. Enzymes are excellent targets for drug development such that a small-molecule enzyme inhibitor can bind to an active or allosteric site to shut down unfavorable enzymatic activities [5]. For enzymes with multiple substrates, however, the loss of all activity can lead to unwanted, on-target side effects. Substrates for gamma-secretase, for example, include the amyloid precursor protein (APP) and NOTCH [6]. The small-molecule gamma-secretase inhibitor semagacestat was developed
to prevent beta-amyloid production in the brain by inhibiting APP proteolysis; yet, clinical trials revealed its on-target side effects likely stemming from semagacestat regulation of NOTCH [7].
reprogram its substrate selectivity, uncovering a new mechanistic concept in small-molecule inhibition that could lead to the development of substrateselective drugs.
Substrate-selective inhibitors have eluded the pharmaceutical industry because high-throughput assays designed to screen for new enzyme inhibitors are not designed to differentiate between natural substrates. Recent work from the Seeliger and Liu laboratories report an innovative solution to this problem by thinking outside of the box, in this case outside of active sites, to develop substrate-selective inhibitors that operate by binding to exo sites [8]. Exo sites are small-molecule binding pockets that do not overlap with an enzyme’s active site (where the chemistry takes place) (Figure 1). Their work demonstrates that occupation of an exo site with a small molecule can reshape the surface of an enzyme to
Earlier work from this group identified the first physiologically active IDE inhibitor (6bK) from a DNA-encoded library (DEL) selection [9]. Unlike other nonspecific IDE inhibitors, which also target other related metalloproteases, 6bK proved to be incredibly selective for IDE. Rigorous biochemical and structural studies revealed that 6bK owed its selectivity to its binding of an exo site on IDE, as opposed to its conventional catalytic site. By contrast, the active sites of closely related enzymes are highly conserved to maintain catalytic activity and, as a result, active-site inhibitors often inhibit related enzymes. Using 6bK to investigate IDE biochemistry and biology, Maianti et al.
Trends in Pharmacological Sciences, October 2019, Vol. 40, No. 10
717
Trends in Pharmacological Sciences
revealed that in addition to insulin, glucagon and amylin are also endogenous IDE substrates [9]. Specifically, upon an oral glucose challenge, IDE primarily regulates insulin, but upon intraperitoneal glucose challenge, IDE mostly regulates glucagon, which leads to elevated glucose levels. Thus, IDE represents an ideal case to study substrate-selective inhibition. In particular, the selective inhibition of insulin degradation, but not glucagon degradation, could be of therapeutic benefit. By selectively binding to an exo site, 6bK raises the possibility that IDE inhibitors could selectively preclude binding of the larger substrates, such as insulin, while permitting the binding and processing of the smaller substrates, such as glucagon. Maianti and coworkers worked towards this goal by developing a highthroughput assay for exo-site binders by utilizing a fluorescent derivative of 6bK to screen for candidates that displaced this ligand from IDE [8]. An initial screen with 9598 structurally diverse compounds followed by an expanded screen with a more targeted chemical library led to the identification of 12 potential inhibitors. A secondary screen comparing insulin and glucagon degradation winnowed this number down to two compounds that preferentially inhibited insulin degradation while only partially effecting glucagon proteolysis. Medicinal chemistry on one of these scaffolds led to the identification of two remarkable compounds that could completely inhibit insulin degradation while only reducing glucagon-degrading activity modestly, even at the highest concentrations of inhibitor. Furthermore, these compounds were incredibly specific, with 10 000-fold selectivity for IDE over similar metallopeptidases. Structural studies explained the substrate selectivity and re-
718
maining enzyme activity. When bound to the exo site, the inhibitors overlapped with insulin but not glucagon binding sites. Furthermore, the structures of inhibitor-bound IDE and IDE alone were identical, demonstrating that these inhibitors did not change the IDE structure or activity, enabling IDE to continue to cleave glucagon while the inhibitor was bound. Together, the data indicate that substrate-selective IDE inhibitors bind to the exo site and, in doing so, reprogram IDE from an insulin and glucagon-degrading enzyme to an enzyme that only processes glucagon. While this work is focused on IDE, the results represent a conceptual advance by demonstrating that it is possible to obtain substrate-selective small-molecule enzyme inhibitors. This can revitalize interest in studying targets that were avoided due to having multiple substrates. Moreover, this approach should be applicable to other enzymes with exo sites that partake in substrate binding, which likely includes other protein-modifying enzymes such as proteases and kinases. By thinking outside of the typical active or allosteric site modes of inhibition, Maianti and colleagues illuminate the importance of exo sites in substrate selectivity by elucidating a structure–function relationship associated with exo-site inhibition [8,9]. 1The
Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA *Correspondence: [email protected] https://doi.org/10.1016/j.tips.2019.07.009
3.
4.
5.
6.
7. 8.
9.
restoration of metabolic balance. Mol. Cell 66, 789–800 Yen, H.C. and Elledge, S.J. (2008) Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science 322, 923–929 Julien, O. et al. (2016) Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. Proc. Natl. Acad. Sci. U. S. A. 113, E2001–E2010 Copeland, R.A. (2005) Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem. Anal. 46, 1–265 Zhang, Z. et al. (2000) Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat. Cell Biol. 2, 463–465 Doody, R.S. et al. (2013) A phase 3 trial of semagacestat for treatment of Alzheimer’s disease. N. Engl. J. Med. 369, 341–350 Maianti, J.P. et al. (2019) Substrate-selective inhibitors that reprogram the activity of insulin-degrading enzyme. Nat. Chem. Biol. 15, 565–574 Maianti, J.P. et al. (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature 511, 94–98
Spotlight
The Sodium Channel Voltage Sensor Slides to Rest Vladimir Yarov-Yarovoy1,* and Paul DeCaen2,* Voltage-gated sodium channels (Navs) initiate the action potential waveforms in excitable cells. The molecular mechanisms controlling this process have been actively debated. New prokaryotic Nav structures by Wisedchaisri et al. have completed our understanding of the molecular conformations required for cellular electrical signaling, and provide key templates for research to examine eukaryotic Navs.
ª 2019 Elsevier Ltd. All rights reserved.
References 1. Stryer, L. et al. (2019) Biochemistry, Macmillan Learning 2. Garcia, D. and Shaw, R.J. (2017) AMPK: mechanisms of cellular energy sensing and
Trends in Pharmacological Sciences, October 2019, Vol. 40, No. 10
Hodgkin, Huxley, and Katz, 67 years ago, measured the current–voltage relationship in the membrane of the giant axon and concluded that an influx of sodium ions across the membrane was
resolve a long-standing discrepancy between human and mouse immunology, where IL-13 had been known to drive IgE switching in human B cells but not in mouse B cells. Further supporting a role for IL-13, Gowthaman et al. demonstrated that a subset of activated mouse B cells in the germinal center expressed the IL-13 receptor and could be induced to switch to IgE in the presence of IL-13. Gowthaman et al. also observed that high-affinity IgE was significantly higher in T-DOCK8-/- mice, while high-affinity IgG1 was lower compared to that in the control group. As a recent study has shown that high-affinity-IgE-producing plasma cells can differentiate from IgG1+ memory B cells upon reexposure to allergens [9], the loss of high-affinity IgG1 in T-DOCK8-/- mice indicates that Tfh13 cells induce a major population of IgG1+ B cells switching to IgE-secreting B cells. A key question is whether IL-13 produced by Tfh13 cells helps IgG1+ memory B cells switch to IgE-secreting plasma cells. However, whether the switch to IgE occurs from high-affinity IgG1 memory cells was not addressed. If IgG1+ memory B cells display enriched expression of IL-13 receptor, it could provide a mechanism for this selective response. Intriguingly, Gowthaman et al. showed that helminth infections could not induce Tfh13 cells or high-affinity IgE, although strong Th2 responses developed. Thus, Th2 cell responses do not always correlate with Tfh13 cell responses. What are the mechanisms for why helminth infections are different from allergies in terms of induction of Tfh13 cells? Solving this question can help us better understand allergies and may also help to develop specific therapeutics for different Th2-mediated diseases such as asthma and atopic dermatitis.
716
Currently, the only treatment that can prevent the mast cell degranulation response that leads to an anaphylactic shock is nonspecific IgE depletion therapy using omalizumab, a monoclonal antibody that inhibits IgE from binding to mast cells [10]. Treatments that could specifically inhibit the generation of high-affinity IgE could provide an alternative therapeutic strategy to block severe allergic responses and anaphylaxis. Since Tfh13 cells express high levels of GATA3, the use of a GATA3 DNAzyme treatment to downregulate Tfh13 cells and high-affinity IgE may have therapeutic benefit for severe allergies. Gowthaman et al. note that clinical trials of a GATA3 DNAzyme have efficacy in reducing eosinophilia, but it is unknown if this drug could affect Tfh13 or IgE responses. Ultimately, understanding the cellular mechanisms for how B cells are stimulated to produce high-affinity IgE in atopic diseases is a clinically important area of immunology research. Eisenbarth and colleagues have now made a major breakthrough on this important question, revealing how a new T cell population, Tfh13 cells, is critical for high-affinity IgE development. These findings lay the groundwork for a deeper understanding of the regulation of high-affinity IgE and anaphylaxis, and provide the basis for precision immunotherapy of atopic diseases. 1Department
of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA *Correspondence: [email protected] https://doi.org/10.1016/j.tips.2019.08.007 ª 2019 Published by Elsevier Ltd.
2. Asher, M.I. et al. (2006) Worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat multicountry crosssectional surveys. Lancet 368, 733–743 3. Iweala, O.I. and Burks, A.W. (2016) Food allergy: our evolving understanding of its pathogenesis, prevention, and treatment. Curr. Allergy Asthma Rep. 16, 37 4. Blank, U. and Rivera, J. (2006) Assays for regulated exocytosis of mast cell granules. Curr. Protoc. Cell Biol. 15, 11 5. Kobayashi, T. et al. (2017) Follicular helper T cells mediate IgE antibody response to airborne allergens. J. Allergy Clin. Immunol. 139, 300–313 6. Xiong, H. et al. (2012) Sequential class switching is required for the generation of high affinity IgE antibodies. J. Exp. Med. 209, 353–364 7. Gowthaman, U. et al. (2019) Identification of a T follicular helper cell subset that drives anaphylactic IgE. Science. Published online August 1, 2019. https://doi.org/10.1126/ science.aaw6433 8. Vinuesa, C.G. et al. (2016) Follicular helper T cells. Annu. Rev. Immunol. 34, 335–368 9. He, J.S. et al. (2017) IgG1 memory B cells keep the memory of IgE responses. Nat. Commun. 8, 641 10. Kawakami, T. and Blank, U. (2016) From IgE to omalizumab. J. Immunol. 197, 4187–4192
Spotlight
Substrate-Selective Enzyme Inhibitors Qian Chu,1 Tina Chang,1 and Alan Saghatelian1,* Enzymes with multiple substrates pose a unique challenge for drug development because of an increased potential for on-target side effects. Maianti and colleagues (Nat. Chem. Biol., 2019) identify novel exo-site inhibitors with abilities to alter the substrate-selectivity of insulindegrading enzymes (IDE). Their work illuminates new therapeutic avenues for discovering small-molecule enzyme inhibitors and redefines our current understanding of drugging enzymes with multiple substrates.
References 1. Sicherer, S.H. and Sampson, H.A. (2007) Peanut allergy: emerging concepts and approaches for an apparent epidemic. J. Allergy Clin. Immunol. 120, 491–503
Trends in Pharmacological Sciences, October 2019, Vol. 40, No. 10
Biochemistry textbooks often depict biochemical pathways as a one-to-one relationship between an enzyme and a
Trends in Pharmacological Sciences
Figure 1. Comparison of Exo-Site Inhibitors to Active-Site Inhibitors. Many enzymes contain exo sites, small-molecule binding sites that do not overlap with the active site, that serve as a binding site for substrates. Activesite inhibitors (orange, right side) block all substrate turnover and, in the case of enzymes with multiple substrates (black, red substrates), this can lead to on-target side effects (inhibition of the red substrate). By contrast, exo-site inhibitors (blue, left) can reprogram an enzyme to become substrate selective, inhibiting the processing of the beneficial black substrate while allowing enzymatic processing of the deleterious red substrate, to mitigate side effects. By demonstrating the feasibility of developing substrate-selective exo-site inhibitors, Maianti and colleagues introduce a new concept into the drug discovery toolbox.
substrate [1]. While this may be true for enzymes involved in small molecule metabolism, protein-modifying enzymes (e.g., proteases, ubiquitin ligases, and kinases) almost always have multiple substrates [2–4]. In these systems, biological phenotypes may arise from the coordinated regulation of multiple substrates, with different substrates contributing to distinct cellular or physiological pathways. Enzymes are excellent targets for drug development such that a small-molecule enzyme inhibitor can bind to an active or allosteric site to shut down unfavorable enzymatic activities [5]. For enzymes with multiple substrates, however, the loss of all activity can lead to unwanted, on-target side effects. Substrates for gamma-secretase, for example, include the amyloid precursor protein (APP) and NOTCH [6]. The small-molecule gamma-secretase inhibitor semagacestat was developed
to prevent beta-amyloid production in the brain by inhibiting APP proteolysis; yet, clinical trials revealed its on-target side effects likely stemming from semagacestat regulation of NOTCH [7].
reprogram its substrate selectivity, uncovering a new mechanistic concept in small-molecule inhibition that could lead to the development of substrateselective drugs.
Substrate-selective inhibitors have eluded the pharmaceutical industry because high-throughput assays designed to screen for new enzyme inhibitors are not designed to differentiate between natural substrates. Recent work from the Seeliger and Liu laboratories report an innovative solution to this problem by thinking outside of the box, in this case outside of active sites, to develop substrate-selective inhibitors that operate by binding to exo sites [8]. Exo sites are small-molecule binding pockets that do not overlap with an enzyme’s active site (where the chemistry takes place) (Figure 1). Their work demonstrates that occupation of an exo site with a small molecule can reshape the surface of an enzyme to
Earlier work from this group identified the first physiologically active IDE inhibitor (6bK) from a DNA-encoded library (DEL) selection [9]. Unlike other nonspecific IDE inhibitors, which also target other related metalloproteases, 6bK proved to be incredibly selective for IDE. Rigorous biochemical and structural studies revealed that 6bK owed its selectivity to its binding of an exo site on IDE, as opposed to its conventional catalytic site. By contrast, the active sites of closely related enzymes are highly conserved to maintain catalytic activity and, as a result, active-site inhibitors often inhibit related enzymes. Using 6bK to investigate IDE biochemistry and biology, Maianti et al.
Trends in Pharmacological Sciences, October 2019, Vol. 40, No. 10
717
Trends in Pharmacological Sciences
revealed that in addition to insulin, glucagon and amylin are also endogenous IDE substrates [9]. Specifically, upon an oral glucose challenge, IDE primarily regulates insulin, but upon intraperitoneal glucose challenge, IDE mostly regulates glucagon, which leads to elevated glucose levels. Thus, IDE represents an ideal case to study substrate-selective inhibition. In particular, the selective inhibition of insulin degradation, but not glucagon degradation, could be of therapeutic benefit. By selectively binding to an exo site, 6bK raises the possibility that IDE inhibitors could selectively preclude binding of the larger substrates, such as insulin, while permitting the binding and processing of the smaller substrates, such as glucagon. Maianti and coworkers worked towards this goal by developing a highthroughput assay for exo-site binders by utilizing a fluorescent derivative of 6bK to screen for candidates that displaced this ligand from IDE [8]. An initial screen with 9598 structurally diverse compounds followed by an expanded screen with a more targeted chemical library led to the identification of 12 potential inhibitors. A secondary screen comparing insulin and glucagon degradation winnowed this number down to two compounds that preferentially inhibited insulin degradation while only partially effecting glucagon proteolysis. Medicinal chemistry on one of these scaffolds led to the identification of two remarkable compounds that could completely inhibit insulin degradation while only reducing glucagon-degrading activity modestly, even at the highest concentrations of inhibitor. Furthermore, these compounds were incredibly specific, with 10 000-fold selectivity for IDE over similar metallopeptidases. Structural studies explained the substrate selectivity and re-
718
maining enzyme activity. When bound to the exo site, the inhibitors overlapped with insulin but not glucagon binding sites. Furthermore, the structures of inhibitor-bound IDE and IDE alone were identical, demonstrating that these inhibitors did not change the IDE structure or activity, enabling IDE to continue to cleave glucagon while the inhibitor was bound. Together, the data indicate that substrate-selective IDE inhibitors bind to the exo site and, in doing so, reprogram IDE from an insulin and glucagon-degrading enzyme to an enzyme that only processes glucagon. While this work is focused on IDE, the results represent a conceptual advance by demonstrating that it is possible to obtain substrate-selective small-molecule enzyme inhibitors. This can revitalize interest in studying targets that were avoided due to having multiple substrates. Moreover, this approach should be applicable to other enzymes with exo sites that partake in substrate binding, which likely includes other protein-modifying enzymes such as proteases and kinases. By thinking outside of the typical active or allosteric site modes of inhibition, Maianti and colleagues illuminate the importance of exo sites in substrate selectivity by elucidating a structure–function relationship associated with exo-site inhibition [8,9]. 1The
Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology, 10010 N. Torrey Pines Rd, La Jolla, CA 92037, USA *Correspondence: [email protected] https://doi.org/10.1016/j.tips.2019.07.009
3.
4.
5.
6.
7. 8.
9.
restoration of metabolic balance. Mol. Cell 66, 789–800 Yen, H.C. and Elledge, S.J. (2008) Identification of SCF ubiquitin ligase substrates by global protein stability profiling. Science 322, 923–929 Julien, O. et al. (2016) Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. Proc. Natl. Acad. Sci. U. S. A. 113, E2001–E2010 Copeland, R.A. (2005) Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem. Anal. 46, 1–265 Zhang, Z. et al. (2000) Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat. Cell Biol. 2, 463–465 Doody, R.S. et al. (2013) A phase 3 trial of semagacestat for treatment of Alzheimer’s disease. N. Engl. J. Med. 369, 341–350 Maianti, J.P. et al. (2019) Substrate-selective inhibitors that reprogram the activity of insulin-degrading enzyme. Nat. Chem. Biol. 15, 565–574 Maianti, J.P. et al. (2014) Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature 511, 94–98
Spotlight
The Sodium Channel Voltage Sensor Slides to Rest Vladimir Yarov-Yarovoy1,* and Paul DeCaen2,* Voltage-gated sodium channels (Navs) initiate the action potential waveforms in excitable cells. The molecular mechanisms controlling this process have been actively debated. New prokaryotic Nav structures by Wisedchaisri et al. have completed our understanding of the molecular conformations required for cellular electrical signaling, and provide key templates for research to examine eukaryotic Navs.
ª 2019 Elsevier Ltd. All rights reserved.
References 1. Stryer, L. et al. (2019) Biochemistry, Macmillan Learning 2. Garcia, D. and Shaw, R.J. (2017) AMPK: mechanisms of cellular energy sensing and
Trends in Pharmacological Sciences, October 2019, Vol. 40, No. 10
Hodgkin, Huxley, and Katz, 67 years ago, measured the current–voltage relationship in the membrane of the giant axon and concluded that an influx of sodium ions across the membrane was