Development of Novel Nuclease Inhibitors as Potential Anticancer Agents
Lead Research Organisation:
University of Oxford
Abstract
This project falls within the EPSRC Chemical biology and biological chemistry research area.
The main aim of the project is to synthesise potent SNM1A/B nuclease inhibitors, whilst employing mechanistic and structural information, as well as the outputs from screening. The obtained results may lead to the development of novel inhibitors with ability to be used as potential anticancer agents.
SNM1A and SNM1B are exonucleases and the members of the human Metallo-b-lactamase (MBL) fold protein family. MBL-fold proteins were first observed in bacteria and are primarily known for their function in the antibacterial resistance. However, at least 18 human MBL-fold proteins that contain conserved motifs in their active site similar to those observed in bacterial MBLs have been identified.
An important function of SNM1A/B is to repair the damaged DNA with interstrand cross-links (ICLs), which is responsible for causing genomic instability. ICLs can be induced by important chemotherapeutic agents such as cis-platin. Knocking down the SNM1A/B in cells results in increased sensitivity to ICLs-inducing therapeutics. Therefore, inhibiting those nucleases has potential to increase effectiveness of cancer chemotherapy.
Chemical similarities between the bacterial MBLs and SNM1A/B proteins have been already observed. b-Lactam antibiotics, which are targeted in resistance mechanism by bacterial MBLs, have been screened against SNM1A/B. It has been reported that certain cephalosporins (i.e. cefotaxime and ceftriaxone) act as reversible inhibitors with uM potency for both SNM1A/B exonucleases. There are already starting points for the development of inhibitors both from cephalosporin based inhibitors and a high-throughput screen (HTS). The following steps will be undertaken: firstly, compounds from the HTS, as well as set of close analogues will be synthesised for structure activity relationship (SAR) studies. Simultaneously, compounds known to inhibit bacterial MBLs will be screened - although the bacterial and human MBL fold enzymes have very different function, at the same time they also have highly conserved active site. Furthermore, project will also include the design of high energy intermediate analogues. Their application has been promising in case of bacterial MBLs, in particular bicyclic boronate inhibitors. Thus, boronate analogous of the phosphate linkage in nucleic acids will be synthesised.
For screening of both compound libraries and newly synthesised compounds a number of assays are proposed to be performed. Those assays have been developed by the Schofield group together with collaborators and used for characterization of SNM1A/B nucleases, as well as the previous screens. In vitro assays that are proposed for this project will include both biophysical and biochemical assays: thermal (TM) shift assay; surface plasmon resonance (SPR) assay; 1H NMR binding assay; X-ray crystallography assay; real-time fluorescence assay; and gel-based assay. Further cell based assay for in vivo activity can be attempted. The use of those different types of assays will allow both achieving more conclusive results and potential detection of false positives. Additionally, the assays will provide information about binding, potency and SAR of the screened small molecules. Consequently, it will allow us to design new inhibitors.
The ultimate goal is to develop novel drugs that would be used in cancer combination therapy with ICLs-inducing agents. Additionally, the developed inhibitors might assist in designing chemical probes that could be used for further characterisation of the function of SNM1A/B nucleases.
The main aim of the project is to synthesise potent SNM1A/B nuclease inhibitors, whilst employing mechanistic and structural information, as well as the outputs from screening. The obtained results may lead to the development of novel inhibitors with ability to be used as potential anticancer agents.
SNM1A and SNM1B are exonucleases and the members of the human Metallo-b-lactamase (MBL) fold protein family. MBL-fold proteins were first observed in bacteria and are primarily known for their function in the antibacterial resistance. However, at least 18 human MBL-fold proteins that contain conserved motifs in their active site similar to those observed in bacterial MBLs have been identified.
An important function of SNM1A/B is to repair the damaged DNA with interstrand cross-links (ICLs), which is responsible for causing genomic instability. ICLs can be induced by important chemotherapeutic agents such as cis-platin. Knocking down the SNM1A/B in cells results in increased sensitivity to ICLs-inducing therapeutics. Therefore, inhibiting those nucleases has potential to increase effectiveness of cancer chemotherapy.
Chemical similarities between the bacterial MBLs and SNM1A/B proteins have been already observed. b-Lactam antibiotics, which are targeted in resistance mechanism by bacterial MBLs, have been screened against SNM1A/B. It has been reported that certain cephalosporins (i.e. cefotaxime and ceftriaxone) act as reversible inhibitors with uM potency for both SNM1A/B exonucleases. There are already starting points for the development of inhibitors both from cephalosporin based inhibitors and a high-throughput screen (HTS). The following steps will be undertaken: firstly, compounds from the HTS, as well as set of close analogues will be synthesised for structure activity relationship (SAR) studies. Simultaneously, compounds known to inhibit bacterial MBLs will be screened - although the bacterial and human MBL fold enzymes have very different function, at the same time they also have highly conserved active site. Furthermore, project will also include the design of high energy intermediate analogues. Their application has been promising in case of bacterial MBLs, in particular bicyclic boronate inhibitors. Thus, boronate analogous of the phosphate linkage in nucleic acids will be synthesised.
For screening of both compound libraries and newly synthesised compounds a number of assays are proposed to be performed. Those assays have been developed by the Schofield group together with collaborators and used for characterization of SNM1A/B nucleases, as well as the previous screens. In vitro assays that are proposed for this project will include both biophysical and biochemical assays: thermal (TM) shift assay; surface plasmon resonance (SPR) assay; 1H NMR binding assay; X-ray crystallography assay; real-time fluorescence assay; and gel-based assay. Further cell based assay for in vivo activity can be attempted. The use of those different types of assays will allow both achieving more conclusive results and potential detection of false positives. Additionally, the assays will provide information about binding, potency and SAR of the screened small molecules. Consequently, it will allow us to design new inhibitors.
The ultimate goal is to develop novel drugs that would be used in cancer combination therapy with ICLs-inducing agents. Additionally, the developed inhibitors might assist in designing chemical probes that could be used for further characterisation of the function of SNM1A/B nucleases.
Organisations
| Description | Discussion and Q&A with year 12th students visiting Chemistry Department |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | The meeting was organised as part of trip for the students was part of outreach by Department of Chemistry. I was taking part with in a group that included two more DPhil students and one Postdoc. The overall aim of the discussion was to introduce the students to how a research at the university looks like. There had been a lot of interesting questions from the students from anything between "Why we decided to do PhD?" to "Whether any of as had experiments that would contradict any laws of science?". Thanks to the fact that there were 4 of us, each question could get multiple answers depending on the background of particular person. Overall it was a very positive experience and hopefully it was inspiring to the students taking part in it. Only thing I didn't like about it was the way the room was organised as the students where siting in number of rows and most of the questions came from the first 2 rows. It might be possible that the students siting further back felt a more intimidated to ask questions due to the distance and a crowd of people between them and us. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Oxford Chemistry Open Day |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Schools |
| Results and Impact | This year I took part in Open day organised by Department of Chemistry, which took place in Chemistry Teaching Laboratory. This event was a great opportunity for prospective undergraduate students and their parent to see our facilities and find out more about studying Chemistry at Oxford. I was part of group of about 10 students representing Department of Chemistry, which mainly consisted of recent Part II students and DPhil students. I had opportunity to take the students for a tour around Chemistry Teaching Laboratory as this is were the students will be doing all their practical work in first 3 years of chemistry course and as part of the trip I was able to answer all the questions potential students and their families had, this included: "how the course is structured?", "Which college is the best?" or "what are the job perspectives with a degree from chemistry?". For each tour between one and seven students and their families were taken, all dependent on the number of visitors and unoccupied "staff" we had a t a particular time of day. As well I had chance to do one tour around CRL for a group that had a bit more time. There was a broad range of students attending, there were students, who were sure that chemistry is what they wanted to study and there were students still didn't decide between doing physics or chemistry. It was very interesting day and a great opportunity for me to share my own experience of doing undergraduate chemistry at Oxford and all my knowledge about the course. I hope that it was very informative to perspective students and especially for those who were undecided about studying chemistry at Oxford. The only sad view of the day were students who were uninterested with visiting Oxford and sometimes it seemed like it were their parents who decided to take them over for open days. In my opinion it should the students decision on what and where they will study as every one should have their own private experience of University. |
| Year(s) Of Engagement Activity | 2019 |