Project title: Development of orphan G-protein coupled receptor peptidic ligand identification system
Funding: Measure 1.1.1.1 “Industry-Driven Research” of specific objective 1.1.1 “To increase the research and innovation capacity of scientific institutions of Latvia and their ability to attract external funding by investing in human resources and infrastructure”
Project No: 1.1.1.1/16/A/055
Period: 1 January 2017 – 31 December 2019
Project costs:285 245,84 EUR
Principle Investigator: Dr. biol. D. Fridmanis
Summary:
GPCRs are one of the largest membrane protein families in human. The main purpose of these proteins is to detect signals in extracellular environment and transduce them to interior of the cell, thus regulating wide variety of physiological processes. Therefore they are the most intensively studied targets in drug development. According to data provided by “IUPHAR/BPS” more than 90 human GPCRs are without known ligand thus these receptors are termed orphan. However knock-out studies in mice have demonstrated that compounds, targeted to these receptors, could be used in treatment of various diseases. In addition, since various, to mammalians distantly related, organisms are grown commercially worldwide; the number of pharmacy industry relevant orphan receptors could be even greater. In some cases these unknown ligands from other species may even display pharmacological activity in mammalians, thus providing the base for development of novel drugs.
The aim of this Project is to develop humanized yeast S.cerevisiae based GPCR peptide ligand identification system and to evaluate its effectiveness by de-orphanization of C.intestinalis orexin-like receptor and determination of novel ligand’s pharmacological properties. The results of this Project shall provide instrument for the development of novel pharmaceutical compounds and promote development of biopharmaceutical industry in Latvia
Information published 02.01.2017.
Progress of the project
1 January 2017 – 31 March 2017
Experimental development
According to plan the first three months of the project were devoted to creation of double expression plasmid. Since p426GPD and p426TEF expression plasmids that have been successfully employed by our group are nearly identical, then within the scope of these activities we altered the multi-clonal sites of the expression loci in a way that each would contain only two unique cloning/restriction sites, thus becoming mutually compatible. Following the modification of both plasmids we performed the sequencing analysis of both expression loci to ascertain the conformity of acquired sequences to planned ones and carried out the transfer of TEF containing locus from p426TEF to p426GPD plasmid. At the moment we are performing the sequencing analysis of thus acquired construct.
Information published 31.03.2017.
Progress of the project
1 April 2017 – 30 June 2017
Experimental development
During this project phase the functional regions of TEF promoter containing plasmid that were relevant for gene expression were transferred to GDP promoter containing plasmid. Further within thus acquired double expression plasmid MC4R and a-MSH coding genes were inserted and following their insertion conformity of acquired results with the plans was established by sequencing analysis. After acquisition of expression constructs we commenced with evaluation of its functionality.
Information published 30.06.2017.
Progress of the project
1 July 2017 – 30 September 2017
Experimental development
During this project phase we initiated the functional testing of previously created double expression plasmid. Within the scope of these activities the newly created plasmid was inserted into the cells of humanized yeast S.cerevisiae strain MMY28, which were subsequently seeded within the selective media containing petri dish. The media within selected dishes was supplemented with MC4R ligand – a-MSH as the means of positive control. Acquired results were ambiguous therefore experiments shall be repeated during further project stages.
Information published 29.09.2017.
Progress of the project
1 October 2017 – 31 December 2017
Experimental development
During this stage of the project we continued with the evaluation of previously created double expression. During these activities plasmids were repeated transformed within yeast S.cerevisiae strain MMY28 cells, which subsequently were seeded on peri-dishes with selective media that in several cases was supplemented with a-MSH. Acquired results, unfortunately, were again ambiguous, therefore it was decided to assess the expression activity of the plasmids employing Western-Blot with a‑MSH specific antibodies. In parallel to facilitate the progress of the whole project we developed an experimental plan that involved successive yeast S.cerevisiae strain MMY28 cell transformation with two separate plasmids.
Information published 02.01.2018.
Progress of the project
1 January 2018 – 31 March 2018
Experimental development
Within the scope of Experimental development activity experiments on evaluation previously created plasmid ability to secrete a-MSH were re carried out. These experiments involved employment of Western-Blot spotting with a-MSH specific antibody. Acquired results confirmed that a-MSH is being successfully secreted, however, since the signal was rather week, it was concluded that its amount could be insufficient to effectively activate the co-expressed MC4R, which could explain ambiguity in previously acquired results. To resolve this issue, an in-depth literature analysis was carried out and as the result it was concluded that it might be necessary to insert the linker peptide between the secretion signal and secreted peptide. T the moment the creation of this construct is being carried out. In parallel with these activities, an experiment on sequential transformation of two separate (first receptor and then random peptide) plasmids into the yeast S.cerevisiae MMY28 strain cells and their subsequent the culturing in histidine lacking media, that promotes propagation of only those cells that expressed receptor is activated. Acquired results were promising, because cells cultures that contained both plasmids were growing faster that thos that contained only the receptor expression plasmid.
System validation
Since the results that were gained within the scope of “Experimental development activity” displayed that the developed experimental system could be operational, within the scope of this activity we attempted to assess it. During these experiments we created plasmid that instead of a-MSH coding sequence contained coding sequence for randomized peptide. Further this plasmid along with MC4R coding sequence containing plasmid were successively transformed into yeast S.cerevisiae MMY28 strain cells, then cultured for several days within the histidine lacking media and extracted. Further, thus acquired plasmid DNA along with randomized source plasmid were used for generation of randomized region Next Generation Sequencing libraries and sequenced using IonTorrent PGM sequencing system. Acquired results showed that selection of clones which contained a-MSH coding sequence has taken place during the randomization and not culturing stage. Therefore during the next project stages improvements in randomization process shall be made.
Information published 29.03.2018.
Progress of the project
1 April 2018 – 30 June 2018
Experimental development
During this period an expression plasmid that contained linker between secretion signal and peptide. In next project periods experiments shall be carried out to evaluate its peptide secretion efficiency. In addition a new strategy was developed for replacement of a-MSH coding sequence with randomized peptide coding sequence and first experiments were initiated to evaluate the system’s efficiency.
System validation
Although the previous validation experiments displayed significant drawbacks in randomization, they resulted in acquisition of rather large data set. Therefore during this project period in-depth data analysis was performed. Acquired results showed that within data set, which was gained after yeast culturing, the proportion of sequences that coded melanocortin receptor pharmacophore was slightly higher than within dataset, which was gained from randomized plasmid, thus indicating that even in the case of suboptimal randomization the system is working and selection of specific clones in taking place.
Information published: 29.06.2018.
Progress of the project
1 July 2018 – 30 September 2018
Experimental development
During this project period, experiments for testing of the new randomization strategy were performed. Within their framework whole plasmid amplification, employing newly created primer pairs, followed by ligation and transformation were performed. However, in the end, only a small number of plasmid-positive colonies were obtained. However, although these results were not promising following cultivation, next generation sequencing of the randomized plasmid and subsequent data analysis was performed. As a result, it was uncoveredthat the randomization process was essentially successful, as the acquired reads didn’t contain a-MSH coding sequence. Therefore, in order to increase the number of positive colonies of plasmid, a further improvement of the randomization process, which increased the number of colonies by 10 fold, was performed. However, this result also didno’t reach the required randomization level. For this reason, further improvement attempts have been initiated, the result of which will be identified at the next stage of the project.
System validation
During this project phase system validation experiments were performed using the most successfully randomized plasmid that was created during the Experimental Development activity. Thus following a sequential transformation with MC4R and randomized plasmids yeast cells were cultured for two weeks in environment without histidine. After completion of the experiment, the selected plasmid was isolated from culture and currently its sequencing analysis is performed.
Information published: 28.09.2018.
Progress of the project
1 October 2018 – 31 December 2018
Experimental development
At the beginning of this project period, a method for obtaining a randomized plasmid was developed It included employment of DNA oligonucleotide with first six 5 ‘end nucleotides replaced to ribonucleotides. Within the framework of these works, a complete plasmid amplification reaction was performed and the resulting product was treated with RNase H, which specifically cleaves only the RNA within double-stranded RNA-DNA hybrid, thus creating a 3’ overhang. After acquisition of thus created plasmid, it was transformed into E. coli cells and seeded on a petri dish. Unfortunately, the efficiency of plasmid randomization did not differ significantly from the effectiveness of the initial attempts. Therefore, an in-depth analysis of the results were carried out and it was concluded that the oligonucleotide RNA part is subject to auto degradation due to temperature changes during amplification, which prevents the formation of overhang and reduces the efficiency of the fragment end binding. Therefore, in the second part of the project period, an alternative randomization strategy was developed, which included the replacement of deoxythymidine, which is located close to 5 ‘end with deoxyuridine and following treatment with UNG.
System validation
During this phase of the project, an analysis of previously acquired sequencing results was carried. The obtained results showed that in essence the ligand selection system is functional, since the sequence reads that were obtained after cultivation contained the a-MSH coding sequence, while such were not found in the randomized plasmid reads.
Information published 28.12.2018.
Progress of the project
1 January 2019 – 31 March 2019
Experimental development
During this project period, the first experiments were carried out using randomization oligonucleotides that contained deoxyuracil at their 5 ‘end. However, all the amplification attempts were unsuccessful. Given that most of the previously performed experiments of whole plasmid amplification were successful, it was concluded that the employed randomization primer has a very strong secondary structure that prevents its binding to the plasmid. This assumption was also confirmed by the in-silico simulation of its structure. Therefore, using the same principles, an alternative primer variant was developed whose in-silico simulation did not display any strong secondary structure. Further experiments using this primer will be carried out in the next stages of the project.
System validation
At this stage of the project within the scope of this activity, the process of preparing sequencing libraries was optimized. Thus the yeast plasmid expraction procedure was improved and the randomized region amplification conditions were optimized.
Information published 29.03.2019.
Progress of the project
1 April 2019 – 30 June 2019
Experimental development
During this period, experiments involving employment of alternative deoxyuracil containing primers were completed. However, unfortunately, even in this case, all the amplification attempts were unsuccessful. In order to explain the obtained results, the analysis of scientific literature was carried out, in which the properties of different thermostable polymerases were compared. As a result it was concluded that the activity thus far used enzyme was adversely affected by the deoxyuracil, so all previously developed primer pairs were subjected to repeated plasmid amplification experiments using a specially selected uridine tolerant polymerase. The results of these actions confirmed the correctness of the hypothesis and the products obtained by the first primer pair were treated with uracil-DNA glycosylase to create an abasic site at the position of uracil. According to literature data, when subjected to elevated temperature such site is prone to spontaneous cleavage, resulting in formation of overhangs that contribute to circularization. However, the transformation of the resulting plasmid into E.coli cells did not show the desired increase in the number of colonies, therefore it was concluded that spontaneous cleavage is not efficient enough and it is necessary to promote it by employment of enzymes. Further, in order to achieve the desired result a mixture of USER enzymes was purchased from the company “NEB” and its employment significantly increased the number of transformation positive colonies, thus successfully completing experimental development activity.
System validation
During this period preparation of sequencing libraries was significantly improved and we developed primers that shall enable the employment of BMC’s recently acquired Illumina MiSeq sequencer, which is more accurate sequencing system than previously used IonTorrent PGM device. In addition, the plasmid extraction procedure from yeast cells was also significantly improved, which shall significantly improve the reproducibility of future results.
Information published 28.06.2019.
Progress of the project
1 July 2019 – 30 September 2019
Experimental development
During this period, validation experiments of the entire ligand selection system were carried out. Within the scope of these experiments, an expression plasmid containing the MC4R coding sequence was transformed into humanized S.cerevisiae yeast strain MMY28 cells and, after selection of transformation positive clones, activation experiments were performed to identify three clones with the highest activation efficiency. After identification, these clones were transformed with a plasmid that encodes a random peptide and culture was subjected to selective cultivation for 12 days. Samples for plasmid isolation and randomized region sequencing were collected starting from day 3. The results showed that the proportion of the plasmid encoding the pharmacophore-containing peptide increased significantly (from 0.007% on day 1 to ~ 10% on day 5) and remained constant throughout the remainder of the experiment. Therefore, these results indicate that the developed ligand selection system is functional and will be validated in the subsequent stages of the study with a human MC3R and ascidia orexin-like receptor.
System validation
During this period preparation of sequencing libraries was significantly improved and we developed primers that shall enable the employment of BMC’s recently acquired Illumina MiSeq sequencer, which in comparison to previously used IonTorrent PGM system is characterized by higher accuracy. In addition, the plasmid extraction procedure from yeast cells was also significantly improved, which shall significantly improve the reproducibility of future results.
Information published 30.09.2019.
Progress of the project
1 October 2019 – 31 December 2019
Experimental development
During this phase of the project, validation experiments of the whole breeding system were continued. Within these, expression plasmid containing the MC3R coding sequence was transformed into MMY28 (Gas) and MMY14 (Gaq) cells of humanized S.cerevisiae yeast strains and, after selection of transformation positive colonies, activation experiments were performed on five cultures of each transformation colony to identify three with the highest activation efficiency, but unfortunately no ligand-dependent growth rate alterations were observed. This situation is likely due to the fact that the presence of the receptor was sufficient to activate the intracellular signaling cascades, since the culture density in the positive control wells was identical to that in the other wells. For this reason, work on the MC3R was not continued, but experiments with CIHCR were started. As before, the humanized S.cerevisiae yeast strain MMY28 (Gas) and MMY14 (Gaq), as well as MMY19 (Ga12), MMY20 (Ga13) and MMY20 (Ga14) cells were transformed with CIHCR coding sequence containing expression plasmid and after selection of transformation-positive colonies, five were transformed with a random peptide-encoding plasmid. This was followed by 12-day selective cultivation and starting from day 3 harvested for plasmid isolation and randomized region sequencing. Unfortunately, the results were negative – no differentially represented peptide was found at any time point. In parallel to these works, a manuscript on developed randomization approach was prepared and submitted to the journal “Nucleic Acids Research”.
System validation
Within the framework of this project activity, all intellectual property created during the project was identified and a report was prepared on their commercialization potential.
Information published 30.12.2019.