Background Germline variants affecting the proofreading activity of polymerases epsilon and delta... more Background Germline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases. Methods A training set of 23 variants considered pathogenic or benign was u...
Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for ca... more Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.
Despite their efficacy in preclinical studies, full-length 4-1BB-agonistic IgGs have not advanced... more Despite their efficacy in preclinical studies, full-length 4-1BB-agonistic IgGs have not advanced in clinical development due to their severe hepatotoxicity. In this study, we provide preclinical proof-of-concept for a humanized Fc-free tumor-specific 4-1BBagonistic trimerbody demonstrating anti-tumor activity against a wide range of human tumors in humanized immunoavatar mice, as well as synergy with immune checkpoint blockers. This approach which may provide a way to elicit responses in most cancer patients while avoiding Fc-mediated adverse reactions. These findings demonstrate that EGFR is an effective target for the development of a broadly applicable tumor-specific 4-1BB mediated immunotherapy, and support the development of the Trimerbody ®-EGFR×4-1BB as a clinical candidate for treatment of advanced solid tumors.
MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interac... more MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apopto...
SummaryAuto-phosphorylation controls the transition between discrete functional and conformationa... more SummaryAuto-phosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlaying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is ade factoc-Src auto-phosphorylation site with slow time-resolution kinetics and strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes fast kinetics and a cis-to-trans phosphorylation-switch that controls c-terminal Tyr 530 auto-phosphorylation, enzyme specificity and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular phosphorylation in which a c-terminal palindromic phospho-motif flanking Tyr 530 on the susbtrate molecule engages the P-loop of the active kinase for ready entry prior catalysis. Perturbation of the phospho-motif accounts for c-Src disfunction as indicated by viral and a colorectal...
Acta Crystallographica Section D-biological Crystallography, May 23, 2003
The iron superoxide dismutase from an eukaryote, the leguminous plant Vigna unguiculata, has been... more The iron superoxide dismutase from an eukaryote, the leguminous plant Vigna unguiculata, has been crystallized for the first time. At cryogenic temperatures, monoclinic crystals diffract to a resolution limit of 1.80 Å using synchrotron radiation.
receptor specificity: Apoptosis induction by TRAIL
Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be det... more Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be determined by
The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) ... more The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4...
Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among ce... more Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 Å of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel -sheets and one ␣-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5. * Financial support was obtained through Ministerio de Educació n y Ciencia Grants BFU-2005-02403, GEN2003-20642-C09-02) (to G. M.), BIO2003-02246 (to F. J. B.), SAF2004-3251 (to F. L.) as well as Ministerio de Sanidad y Consumo Grant FIS CP05/100 (to M. R. S.
Acta Crystallographica Section A Foundations of Crystallography, 2005
AlvinFd sub-family is Pseudomonas aeruginosa Fd (PaFd), which also shows the above unusual redox ... more AlvinFd sub-family is Pseudomonas aeruginosa Fd (PaFd), which also shows the above unusual redox properties. The PaFd structure has been determined to 1.32 resolution, the highest up to now for the AlvinFd sub-family. The detailed structural information that this structure affords, i.e. the precise geometry of the [4Fe-4S] clusters combined with the conformation of the surrounding residues and the electrostatic properties of the protein around and between the two clusters, the EPR spectroscopy studies, as well as a thorough comparison with existing high resolution structures of [4Fe-4S]containing proteins, provide structural reasons for the unusual redox properties of the Alvin-like 2[4Fe-4S] Fds.
Improved Tumor Cell Killing by Trail Requires Selective and High Affinity Receptor Activation
Apoptosis can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in ... more Apoptosis can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a wide range of tumor cells, but not in non-transformed cells. TRAIL interaction with receptors DR4 or DR5 induces apoptosis, whereas DcR1, DcR2 and osteoprotegerin are decoy receptors for TRAIL. TRAIL variants (single mutant, D269H, and double mutant D296H/E195R) engineered to selectively activate DR5 but not DR4 enhanced pro-caspase-8 processing and apoptosis in tumor cells, compared to wild-type TRAIL. D269H/E195R showed a 17 fold faster kinetics of receptor activation than wild-type TRAIL. The robust receptor activation was not due to altered 3D ligand structures or aggregation but to high affinity and selectivity towards DR5, which prevented ligand-induced receptor heteromerization and neutralization by decoys. These findings have important implications for the design of anticancer therapeutic agents and cytokine engineering.
Acta Crystallographica Section A Foundations of Crystallography, 2005
The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the ... more The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the photoreceptor proteins using a flavin chromophore, where the other two families are phototropins and cryptochromes. We have determined the crystal structure of the Tll0078 protein from Thermosynechococcus elongatus BP-1, which contains a BLUF domain bound to FAD, at 2 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 with cell dimensions of a=89.5 Å, b=109.9 Å, and c=169.9 Å. The asymmetric unit contains 10 monomers of Tll0078 (one decamer). Five Tll0078 monomers are located around the non-crystallographic five-fold axis to form a pentamer, and two pentamers related by twofold noncrystallographic symmetry form a decameric assembly. The monomer consists of two domains, the BLUF domain at the Nterminal region and the C-terminal domain. The overall structure of the BLUF domain consists of a five-stranded mixed-sheet with two helices running parallel to it. The isoalloxazine ring of FAD is accommodated in a pocket formed by several highly-conserved amino acid residues in the BLUF domain.
Publisher American Society for Biochemistry and Molecular Biology
Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the p... more Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published
Background Germline variants affecting the proofreading activity of polymerases epsilon and delta... more Background Germline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases. Methods A training set of 23 variants considered pathogenic or benign was u...
Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for ca... more Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.
Despite their efficacy in preclinical studies, full-length 4-1BB-agonistic IgGs have not advanced... more Despite their efficacy in preclinical studies, full-length 4-1BB-agonistic IgGs have not advanced in clinical development due to their severe hepatotoxicity. In this study, we provide preclinical proof-of-concept for a humanized Fc-free tumor-specific 4-1BBagonistic trimerbody demonstrating anti-tumor activity against a wide range of human tumors in humanized immunoavatar mice, as well as synergy with immune checkpoint blockers. This approach which may provide a way to elicit responses in most cancer patients while avoiding Fc-mediated adverse reactions. These findings demonstrate that EGFR is an effective target for the development of a broadly applicable tumor-specific 4-1BB mediated immunotherapy, and support the development of the Trimerbody ®-EGFR×4-1BB as a clinical candidate for treatment of advanced solid tumors.
MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interac... more MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apopto...
SummaryAuto-phosphorylation controls the transition between discrete functional and conformationa... more SummaryAuto-phosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlaying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is ade factoc-Src auto-phosphorylation site with slow time-resolution kinetics and strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes fast kinetics and a cis-to-trans phosphorylation-switch that controls c-terminal Tyr 530 auto-phosphorylation, enzyme specificity and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular phosphorylation in which a c-terminal palindromic phospho-motif flanking Tyr 530 on the susbtrate molecule engages the P-loop of the active kinase for ready entry prior catalysis. Perturbation of the phospho-motif accounts for c-Src disfunction as indicated by viral and a colorectal...
Acta Crystallographica Section D-biological Crystallography, May 23, 2003
The iron superoxide dismutase from an eukaryote, the leguminous plant Vigna unguiculata, has been... more The iron superoxide dismutase from an eukaryote, the leguminous plant Vigna unguiculata, has been crystallized for the first time. At cryogenic temperatures, monoclinic crystals diffract to a resolution limit of 1.80 Å using synchrotron radiation.
receptor specificity: Apoptosis induction by TRAIL
Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be det... more Kinetics in signal transduction pathways involving promiscuous oligomerizing receptors can be determined by
The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) ... more The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4...
Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among ce... more Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 Å of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel -sheets and one ␣-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5. * Financial support was obtained through Ministerio de Educació n y Ciencia Grants BFU-2005-02403, GEN2003-20642-C09-02) (to G. M.), BIO2003-02246 (to F. J. B.), SAF2004-3251 (to F. L.) as well as Ministerio de Sanidad y Consumo Grant FIS CP05/100 (to M. R. S.
Acta Crystallographica Section A Foundations of Crystallography, 2005
AlvinFd sub-family is Pseudomonas aeruginosa Fd (PaFd), which also shows the above unusual redox ... more AlvinFd sub-family is Pseudomonas aeruginosa Fd (PaFd), which also shows the above unusual redox properties. The PaFd structure has been determined to 1.32 resolution, the highest up to now for the AlvinFd sub-family. The detailed structural information that this structure affords, i.e. the precise geometry of the [4Fe-4S] clusters combined with the conformation of the surrounding residues and the electrostatic properties of the protein around and between the two clusters, the EPR spectroscopy studies, as well as a thorough comparison with existing high resolution structures of [4Fe-4S]containing proteins, provide structural reasons for the unusual redox properties of the Alvin-like 2[4Fe-4S] Fds.
Improved Tumor Cell Killing by Trail Requires Selective and High Affinity Receptor Activation
Apoptosis can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in ... more Apoptosis can be activated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a wide range of tumor cells, but not in non-transformed cells. TRAIL interaction with receptors DR4 or DR5 induces apoptosis, whereas DcR1, DcR2 and osteoprotegerin are decoy receptors for TRAIL. TRAIL variants (single mutant, D269H, and double mutant D296H/E195R) engineered to selectively activate DR5 but not DR4 enhanced pro-caspase-8 processing and apoptosis in tumor cells, compared to wild-type TRAIL. D269H/E195R showed a 17 fold faster kinetics of receptor activation than wild-type TRAIL. The robust receptor activation was not due to altered 3D ligand structures or aggregation but to high affinity and selectivity towards DR5, which prevented ligand-induced receptor heteromerization and neutralization by decoys. These findings have important implications for the design of anticancer therapeutic agents and cytokine engineering.
Acta Crystallographica Section A Foundations of Crystallography, 2005
The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the ... more The sensor proteins for blue-light using the FAD (BLUF) domain belong to the third family of the photoreceptor proteins using a flavin chromophore, where the other two families are phototropins and cryptochromes. We have determined the crystal structure of the Tll0078 protein from Thermosynechococcus elongatus BP-1, which contains a BLUF domain bound to FAD, at 2 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 with cell dimensions of a=89.5 Å, b=109.9 Å, and c=169.9 Å. The asymmetric unit contains 10 monomers of Tll0078 (one decamer). Five Tll0078 monomers are located around the non-crystallographic five-fold axis to form a pentamer, and two pentamers related by twofold noncrystallographic symmetry form a decameric assembly. The monomer consists of two domains, the BLUF domain at the Nterminal region and the C-terminal domain. The overall structure of the BLUF domain consists of a five-stranded mixed-sheet with two helices running parallel to it. The isoalloxazine ring of FAD is accommodated in a pocket formed by several highly-conserved amino acid residues in the BLUF domain.
Publisher American Society for Biochemistry and Molecular Biology
Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the p... more Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published
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