Final Report Summary - GCPII_SYSTEM (Design and development of novel reagents, tools, and techniques targeting human glutamate carboxypeptidases II and III)
The Marie Curie International Reintegration Grant (IRG) within the 7th European Community Framework Program is/was aimed to encourage the return of scientists from the United States back to the European Research Area (ERA). The program thus supports and combines two main complementary objectives: (A) excellence in basic or applied research carried out by a returning scientist; (B) the dissemination of the accumulated knowledge and the development of a network of scientific collaborations within the ERA.
(A): Scientific objectives
Our efforts were focused on addressing fundamental questions pertaining (patho)physiological roles of human glutamate carboxypeptidase II (GCPII) and its orthologs/paralogs in healthy tissues as well as in cancer and neurodegeneration. GCPII is a membrane-zinc metallopeptidase implicated in neurotransmission within the nervous system and the absorption of dietary folates in small intestine. At the same time, physiological functions of GCPII in several major tissues (prostate, kidneys, vasculature of solid tumors) are not known. Similarly, there is a limited understanding of physiological roles of other members of GCPII family in humans and other species. Under pathological conditions, GCPII is exploited as a target for imaging of prostate cancer and the vasculature of solid tumors. Additionally, the brain form of GCPII is targeted in preclinical models of a variety of neuropathologies. Consequently, GCPII represents an attractive target of the biomedical research and our second scientific objective focused on the development of novel ligands targeting GCPII that could provide invaluable tools for basic studies and furthermore serve as a starting point for the translational research.
• The development of novel GCPII-specific ligands was the major part of our efforts within this funding scheme. To identify novel GCPII-specific low-molecular weight compounds we used three complementary approaches including (i) structure-assisted drug design, (ii) in vitro screening campaign, and (iii) in vitro screening. Within the sub-project (i) we determined and deposited 22 X-ray structures of complexes between GCPII and small-molecule inhibitors to the Protein Data Bank and published 5 papers in peer review journals. Our findings offered detailed insight into interactions governing GCPII selectivity towards small molecules that could be used for designing the next generation compounds. To carry out an extensive in vitro screening campaign we developed a novel assay based on fluorescence polarization and screened more than 50,000 compounds from libraries of small organic molecules. Newly identified positive hits were further validated and selected for ensuing SAR studies. The results were published in the Journal of Biomolecular Screening in 2012. Finally, collaboration with S. Constanzi, NIH, USA; and J. Neale, Georgetown University, USA we screened approximately 1 million compounds from the virtual zinc-library and evaluated approximately 100 of them in vitro. The best hits inhibited GCPII in low micromolar range confirming the usability of this methodology for further studies.
In addition to studies dealing with low-molecular weight compounds we set up to develop GCPII-specific macromolecular reagents for in vivo imaging of prostate cancer. To this end, in collaboration with Skerra lab (Technical University, Freising, Germany) we set up to develop a non-immunoglobulin engineered scaffolds based on human lipocalin Lcn2. We were able to isolate and characterize a lipocalin variant (called Anticalin) with a sub-nanomolar affinity for GCPII. Results from this project demonstrate that the Anticalin constitutes a novel and promising GCPII-specific reagent that recognizes the target protein in its native conformation and, consequently, can be used for the development of imaging agents for prostate cancer in the future.
• Physiological functions of GCPII and its paralogs/orthologs were studied in several projects. To dissect a role of GCPII in folate metabolism we carried out detailed structural and biochemical characterization of the wild-type enzyme and its His475Tyr variant. Based on these results and contrary to published data we suggest that the H475Y mutation does not influence GCPII 3-D structure or its hydrolyzing activity and it therefore cannot be directly linked lower folate levels in human plasma. NAALADase L is a close paralog of GCPII, but neither the physiological functions nor structural features of NAALADase L are known at present. By solving the X-ray structure of NAALADase L, we provided the first experimental evidence that it is a zinc-dependent metallopeptidase that possesses previously unrecognized aminopeptidase activity. Our data showed that the primary physiological function of NAALADase L is associated with the final stages of peptide hydrolysis and absorption in the human digestive system. In addition to projects aimed at human GCPII, we were studying GCPII orthologs from several model organisms and human pathogens, including C.elegans D.rerio K.lactis S.mansoni and F.hepatica. The objective of this study is to understand physiological role(s) of GCPII in these species and these projects are in different stages of progress, with the most advanced being characterization of R57a, a GCPII ortholog from C.elegans. Here we produced and purified the recombinant protein, were able to find crystallization conditions producing diffracting crystals, and in collaboration with M. Asahina-Jindrova (Institute of Parasitology, Czech Rep) we generated transgenic worms expressing GFP under the R57a promoter. Biochemical experiments focused on substrate specificity characterization are ongoing.
The potential impact of our findings is twofold: (i) Novel GCPII-specific reagents (both small-molecule inhibitors and macromolecules) will be used as research tools to better understand role(s) of GCPII in pathophysiology. Additionally, they can potentially serve as lead compounds for the development of imaging/therapeutic modalities targeting GCPII; (ii) the “basic research“ area should “simply” expand our knowledge of physiological function(s) of GCPII and its paralogs in humans as well as in different model organisms and human pathogens.
B: Reintegration, knowledge transfer, collaborations
In addition to the scientific output the proposed reintegration objectives were realized via my appointment as a head of the Laboratory of Structural Biology at the Institute of Biotechnology AS CR, with the original contract extended till the end of 2016. The lab is now successfully running and comprises nine co-workers, including five students (pre-graduate and PhD). Within the period 2010 – 2014 I was able to obtain additional funds to support my research from various funding bodies, including the Czech Science Foundation (two research grants; 2012 – 2016 and 2015 – 2017), the prestigious the EMBO Installation Grant (2010 – 2015), the J.E. Purkyne fellowship (2011 – 2014), a grant from the Ministry of Education, Youth, and Sports (2010 – 2012). I established several new collaborations with teams from universities and research institutions within the Czech Republic (7 new collaborative projects) and the EU (2 new collaborative projects). The dissemination activities include 7 oral and 7 poster presentations at 11 conferences and 4 peer-review articles in international journals in the period 2010 – 2014. In relation to the wider, non-scientific audience members of my lab and myself have several presentations for the general public (typically high school students) during annual “Week of Science and Technology” organized by the Academy of Science of the CR. Thanks to the Institutional support I procured a crystallization robot and a CCD camera for the evaluation of crystallization plates in 2011. In the combination with an X-ray generator, this equipment allowed me to set up (and run) a crystallization core lab that is now extensively used several research groups from our Institute as well as whole research campus.
Further information can be found in web pages of the Institute of Biotechnology AS CR (http://www.ibt.cas.cz) or the web pages of our laboratory (http://academy5.avcr.cz/lsb/).
(A): Scientific objectives
Our efforts were focused on addressing fundamental questions pertaining (patho)physiological roles of human glutamate carboxypeptidase II (GCPII) and its orthologs/paralogs in healthy tissues as well as in cancer and neurodegeneration. GCPII is a membrane-zinc metallopeptidase implicated in neurotransmission within the nervous system and the absorption of dietary folates in small intestine. At the same time, physiological functions of GCPII in several major tissues (prostate, kidneys, vasculature of solid tumors) are not known. Similarly, there is a limited understanding of physiological roles of other members of GCPII family in humans and other species. Under pathological conditions, GCPII is exploited as a target for imaging of prostate cancer and the vasculature of solid tumors. Additionally, the brain form of GCPII is targeted in preclinical models of a variety of neuropathologies. Consequently, GCPII represents an attractive target of the biomedical research and our second scientific objective focused on the development of novel ligands targeting GCPII that could provide invaluable tools for basic studies and furthermore serve as a starting point for the translational research.
• The development of novel GCPII-specific ligands was the major part of our efforts within this funding scheme. To identify novel GCPII-specific low-molecular weight compounds we used three complementary approaches including (i) structure-assisted drug design, (ii) in vitro screening campaign, and (iii) in vitro screening. Within the sub-project (i) we determined and deposited 22 X-ray structures of complexes between GCPII and small-molecule inhibitors to the Protein Data Bank and published 5 papers in peer review journals. Our findings offered detailed insight into interactions governing GCPII selectivity towards small molecules that could be used for designing the next generation compounds. To carry out an extensive in vitro screening campaign we developed a novel assay based on fluorescence polarization and screened more than 50,000 compounds from libraries of small organic molecules. Newly identified positive hits were further validated and selected for ensuing SAR studies. The results were published in the Journal of Biomolecular Screening in 2012. Finally, collaboration with S. Constanzi, NIH, USA; and J. Neale, Georgetown University, USA we screened approximately 1 million compounds from the virtual zinc-library and evaluated approximately 100 of them in vitro. The best hits inhibited GCPII in low micromolar range confirming the usability of this methodology for further studies.
In addition to studies dealing with low-molecular weight compounds we set up to develop GCPII-specific macromolecular reagents for in vivo imaging of prostate cancer. To this end, in collaboration with Skerra lab (Technical University, Freising, Germany) we set up to develop a non-immunoglobulin engineered scaffolds based on human lipocalin Lcn2. We were able to isolate and characterize a lipocalin variant (called Anticalin) with a sub-nanomolar affinity for GCPII. Results from this project demonstrate that the Anticalin constitutes a novel and promising GCPII-specific reagent that recognizes the target protein in its native conformation and, consequently, can be used for the development of imaging agents for prostate cancer in the future.
• Physiological functions of GCPII and its paralogs/orthologs were studied in several projects. To dissect a role of GCPII in folate metabolism we carried out detailed structural and biochemical characterization of the wild-type enzyme and its His475Tyr variant. Based on these results and contrary to published data we suggest that the H475Y mutation does not influence GCPII 3-D structure or its hydrolyzing activity and it therefore cannot be directly linked lower folate levels in human plasma. NAALADase L is a close paralog of GCPII, but neither the physiological functions nor structural features of NAALADase L are known at present. By solving the X-ray structure of NAALADase L, we provided the first experimental evidence that it is a zinc-dependent metallopeptidase that possesses previously unrecognized aminopeptidase activity. Our data showed that the primary physiological function of NAALADase L is associated with the final stages of peptide hydrolysis and absorption in the human digestive system. In addition to projects aimed at human GCPII, we were studying GCPII orthologs from several model organisms and human pathogens, including C.elegans D.rerio K.lactis S.mansoni and F.hepatica. The objective of this study is to understand physiological role(s) of GCPII in these species and these projects are in different stages of progress, with the most advanced being characterization of R57a, a GCPII ortholog from C.elegans. Here we produced and purified the recombinant protein, were able to find crystallization conditions producing diffracting crystals, and in collaboration with M. Asahina-Jindrova (Institute of Parasitology, Czech Rep) we generated transgenic worms expressing GFP under the R57a promoter. Biochemical experiments focused on substrate specificity characterization are ongoing.
The potential impact of our findings is twofold: (i) Novel GCPII-specific reagents (both small-molecule inhibitors and macromolecules) will be used as research tools to better understand role(s) of GCPII in pathophysiology. Additionally, they can potentially serve as lead compounds for the development of imaging/therapeutic modalities targeting GCPII; (ii) the “basic research“ area should “simply” expand our knowledge of physiological function(s) of GCPII and its paralogs in humans as well as in different model organisms and human pathogens.
B: Reintegration, knowledge transfer, collaborations
In addition to the scientific output the proposed reintegration objectives were realized via my appointment as a head of the Laboratory of Structural Biology at the Institute of Biotechnology AS CR, with the original contract extended till the end of 2016. The lab is now successfully running and comprises nine co-workers, including five students (pre-graduate and PhD). Within the period 2010 – 2014 I was able to obtain additional funds to support my research from various funding bodies, including the Czech Science Foundation (two research grants; 2012 – 2016 and 2015 – 2017), the prestigious the EMBO Installation Grant (2010 – 2015), the J.E. Purkyne fellowship (2011 – 2014), a grant from the Ministry of Education, Youth, and Sports (2010 – 2012). I established several new collaborations with teams from universities and research institutions within the Czech Republic (7 new collaborative projects) and the EU (2 new collaborative projects). The dissemination activities include 7 oral and 7 poster presentations at 11 conferences and 4 peer-review articles in international journals in the period 2010 – 2014. In relation to the wider, non-scientific audience members of my lab and myself have several presentations for the general public (typically high school students) during annual “Week of Science and Technology” organized by the Academy of Science of the CR. Thanks to the Institutional support I procured a crystallization robot and a CCD camera for the evaluation of crystallization plates in 2011. In the combination with an X-ray generator, this equipment allowed me to set up (and run) a crystallization core lab that is now extensively used several research groups from our Institute as well as whole research campus.
Further information can be found in web pages of the Institute of Biotechnology AS CR (http://www.ibt.cas.cz) or the web pages of our laboratory (http://academy5.avcr.cz/lsb/).