- Title
- Small molecule inhibitors targeting the S100A2-p53 complex for potential treatments of pancreatic cancer
- Creator
- Sun, Jufeng
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2021
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Pancreatic cancer (PC) is one of the deadliest human cancers, with the highly metastatic nature presenting a major challenge for improving patient outcomes. These heterogeneous tumours are composed of cancer cells with differing morphologies and phenotypic profiles, making them extremely difficult to treat as they evade targeted therapies. Current combination chemotherapies for PC are highly toxic and only a fraction of patients respond (~30%), with a 5-year survival of ~7%. As a result, new targeted therapies which are effective against PC are urgently needed. The S100 calcium-binding protein A2 (S100A2) isoform is a promising new target for the treatment of PC, a member of the S100 protein family. This protein is upregulated in pancreatic cancer and other cancer tissues, and is a predictive biomarker of PC: in addition, S100A2 modulates the tumour suppressor p53 by binding with its transactivation domain. In PC, over expression of S100A2 inhibits p53, preventing p53 tumour suppression, resulting in aberrant cancer cell proliferation. Thus S100A2 is regarded as a novel p53 target protein. Pilot work with in vivo models of PC showed an increased volume of tumours derived from S100A2 overexpressing cells, with extensive S100A2 expressing liver metastases. In addition, overexpression of S100A2 was validated in the S100A2 overexpressing tumours. Importantly, S100A2 expression is endogenously very high in the BxPC-3 cell line when compared to the other PC cell lines studied in this work. Based on the above investigations, we hypothesise that inhibitors of S100A2-p53 complex may exert an anti-PC role. This thesis reports on the investigation of the effect of compounds designed to target the S100A2-p53 protein-protein interaction (PPI) against a number of both cancerous and two normal human cell lines. A comprehensive review was compiled on the current state of small molecules inhibitors in PC (Chapter Two). Previous study in this area had discovered leads I (MKH2130, N-(4-((4-(3-(4-(3- methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)piperazin-1-yl)sulfonyl)phenyl)acetamide and II (JA1124, N-(6-((4-bromobenzyl)amino)hexyl)-3,5-bis(trifluoromethyl)benzenesulfonamide) which were predicted to block the interaction between S100A2 and p53 via orthosteric competitive binding for protein-protein interface. The first part of this thesis focused on the modification of lead I. Ten focused libraries were designed, synthesised and evaluated; 78 compounds in total, with subsequent compound cytotoxicity assessment performed against 16 human cancer cell lines including six PC cell lines (MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, HPAC and PANC-1) and two normal cell lines (MCF10A and HPDE), leading to enhanced cytotoxic activity. Initial investigations in this work began with lead I: in order to study the influence of different substituted groups on the benzenesulfonamide moiety, and the phenyl ring, compounds in Library 1 and Library 2 were designed, synthesised and evaluated (Chapter 3). Analysis of the screening results of these compounds revealed that the nature of the substituent groups on the benzenesulfonamide moiety does not impart an observable cytotoxicity difference against PC cell line MiaPaCa-2, and electron effects of substituent groups on the phenyl ring do not significantly affect the activity: additionally, steric large or bulky groups may help to increase the cytotoxicity. In order to further study the influence of different groups on the phenyl ring and different substituent position on both the benzenesulfonamide moiety and the phenyl ring on the activity, compound Libraries 3 and 4 with a CF3 group on the benzenesulfonamide moiety were designed, synthesised and evaluated (Chapter Four). Analysis of the biological data of these compounds (Libraries 3 and 4) indicated that a selection of analogues with large or bulky substituents on the phenyl ring exhibited activity with GI50 values from 3.7-18 μM in six PC lines (MiaPaCa-2, BxPC-3, AsPC-1, Capan-2, HPAC and PANC-1) analysed. In particular, one analogue (4-20: compound 4-20 in Chapter Four) showed significant cytotoxicity with sub-micromolar GI50 values in the range of 0.43-0.68 µM in six PC lines tested. This analogue featured a 4-CF3 functionality on the benzenesulfonamide moiety and a 2-OCH3 moiety on the phenyl ring. Further synthesis was conducted based on these results: in order to compare the cytotoxicity disparity between the cyclic and the non-cyclic analogues of lead I and the influence of different length carbon chains between the triazole and the diamine moiety on the activity, a further five libraries (Libraries 5-9) were designed to contain non-cyclic isosteres of the piperazine moiety (Chapter Five). Varying alkyl chain lengths and substituted phenyl rings were synthesised, resulting in a series of analogues with 75% GI50 values <3.2 µM (0.9-9.0 µM) in the six PC cell lines. Analysis of the biological data of all compounds from Libraries 1-9, the structure-activity relationship (SAR) data for the modification of lead I were summarised. Based on the SAR from Libraries 1-9, a testable hypothesis was generated: 3,5-bis(CF3) groups on the benzene- sulfonamide moiety, non-cyclic isosteres of piperazine moiety, a propyl linker (carbon chains between the triazole and the diamine moiety) and large or bulky groups or 2-substituents on the phenyl ring may contribute to enhance the cytotoxicity of lead I analogues. In order to validate the hypothesis from the SAR of lead I, compound Library 10 was designed to include non-cyclic analogues with different large or bulky groups or 2-substituents on the phenyl ring, synthesized and evaluated (Chapter 6), resulting in a selection of analogues 6-1, 6-3 and 6-4 with large or bulky 4-Ph-Ph, 3,5-bis(CF3)Ph and 1-naphthyl moiety respectively, with 90% exhibiting activity <3.0 µM (0.85-7.3 µM) in six PC lines tested, which was consistent with the generated hypothesis. The second part of this thesis focused on the modification of lead II. Three focused libraries were designed, synthesised and evaluated; 24 compounds in this work. In vitro cytotoxicity assessment was performed against 15 human cancer cell lines including six PC cell lines mentioned above and one normal cell line (MCF10A). On the basis of biological evaluation and iterative design, the modification of lead II also saw improved cytotoxic activity against six PC cell lines tested. Initial studies began with lead II: in order to study the influence of different substituted groups on the phenyl ring and different length carbon chains between two nitrogen atoms on the activity, Library 11 and Library 12 were designed, synthesized and evaluated, resulting in a selection of analogues 7-26 and 7-29-35 showed good to excellent activity with ~85 % GI50 values <3 µM (1.4 to 18 µM) against six PC cell lines examined (Chapter Seven). Analysis of the biological data of compounds in libraries 11 and 12 synthesized in this work and 20 compounds previously prepared by Dr. Joey Ambrus in the McCluskey group, the SAR data for the modification of lead II were summarised. Based on the SAR from libraries 11 and 12, a testable hypothesis was generated: 3,5-bis(CF3) groups on the benzene- sulfonamide moiety and a propyl linker (carbon chains between two nitrogen atoms) improve the activity, and the activity is not significantly affected by either electron or steric effects of substituent groups on the phenyl ring when there is a propyl linker. In order to validate the hypothesis from the SAR of lead II, compound Library 13 was designed to include analogues with different electronic-donating or electronic-withdrawing groups on the phenyl ring and a propyl chain, leading to a selection of analogues 8-1 (4-OCH3Ph), 8-4 (3,4-diClPh), 8-7 (4-ClPh), and 8-11 (4-Cl-3-CF3Ph) showed good to excellent cytotoxicity with >80% GI50 values <3.0 µM (1.2-11 µM) in six PC cell lines tested. This analysis validated the hypothesis generated above (Chapter Eight). Overarching analysis of the biological data of all analogues synthesised in this work revealed majority of the most active analogues expressed enhanced activity in the BxPC-3 cell line. In general, lead I analogues showed 80% GI50 values <4.0 µM (0.48-14 µM), and analogues of lead II with 81% GI50 values <3.0 µM (1.2-13 µM). Of these, 4-20 (1- (3-(4-(2-methoxyphenyl)-1H-1,2,3-triazol-1-yl)propyl)-4-((4-trifluoromethyl)phenyl)sulfonyl)piperazine, GI50 = 0.48 µM) in lead I analogues and 8-11 (N-(3-((4-chloro-3-(trifluoromethyl) benzyl)amino)propyl)-3,5-bis(trifluoromethyl)benzenesulfonamide, GI50 = 1.2 µM) in lead II analogues are the most active against BxPC-3 cell line. These data support the overarching hypothesis that inhibition of S100A2-p53 complex has an anti-PC activity as S100A2 expression is endogenously very high in the BxPC-3 cell line when compared to the other PC cell lines investigated in this study. Molecular modelling docking studies for selected compounds was conducted to compare the SAR and activity analysis of analogues synthesised utilising Molecular Operating Environment (MOE) software platform. In general, docking analysis of the active lead I analogues revealed that the triazoloaryl moieties or the benzenesulfonamide moieties (analogues with 2-OCH3 on the phenyl ring) of the molecules align in the same vicinity and direction with p53 from the right hand of the p53 groove when docked; docking analysis of active lead II analogues suggested N-(arylmethyl)aminoalkyl moieties of these molecules also align in the same region and direction with p53 from the right hand of the p53 groove when docked.
- Subject
- small molecule inhibitors; pancreatic cancer; S100 protein family; heterogeneous tumours
- Identifier
- http://hdl.handle.net/1959.13/1514049
- Identifier
- uon:56812
- Rights
- Copyright 2021 Jufeng Sun
- Language
- eng
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