13 research outputs found

    Identification and development of small molecule therapies for the treatment of acute myeloid leukaemia

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    Acute myeloid leukaemia (AML) is a cancer of the bone marrow in which immature myeloid cells exhibit uncontrolled proliferation and failure to differentiate. The global 5-year overall survival rate for AML ranges between 25 – 35% indicating the urgent need for novel therapeutic alternatives. Current therapies include bone marrow transplant (for a minority of suitable patients), chemotherapy and a handful of small molecule drugs approved for selected patient subgroups. Poor survival rates and limited therapy response are partially attributed to inter- and intra- patient heterogeneity. The concept of personalised medicine has emerged as one potential strategy to overcome the diversity of AML patients. For this approach to succeed novel small molecules with unique anti-leukemic properties must be developed to expand the arsenal of clinical alternatives. The primary objective of the thesis was to identify and develop small molecule therapies for the treatment of AML. In paper I we supported the development of novel small molecules and investigated their cytotoxic properties in AML cell lines. N-heterocyclic carbenes were generated and complexed with silver using a novel multi-step synthetic pathway to produce the putative metallodrugs, NHC-1 and NHC-2. Dose response curves were generated for each of the compounds to determine the IC50 of each compound in the AML cell lines, MOLM-13 and HL-60. Cell death induction was characterised as rapid and associated with apoptotic nuclear morphology. Interestingly, we also observed increased phosphatidylserine expression in HL-60 cells treated with the silver NHC complexes as compared with cytarabine. In paper II we identify the tryptamine derivative, Serdemetan (SDM), as a candidate for small molecule therapy in AML. SDM was originally discovered as part of an internal drug development program at Janssen Pharmaceutical, attempting to identify novel Hdm2 inhibitors for use in solid cancers. In AML cell lines and patient samples SDM activity varied independent of p53 status, suggesting the agent may have additional molecular targets. SDM was well tolerated and significantly prolonged survival in preclinical models of AML including the syngeneic BNML rat model and MOLM-13 xenograft, further indicating the agent’s suitability for development in AML. Interestingly, our studies revealed novel mechanisms of action including upregulation of autophagy markers and depletion of Akt1 expression. In paper III we propose repositioning Hydroxyurea (HU) and Valproic Acid (VPA) as a combination therapy for the treatment of AML. Both agents have previously been trailed clinically in AML and are generally well tolerated. HU and VPA combined to induce synergistic cell death in multiple AML cell lines. Unlike SDM, combination induced apoptosis and proliferation arrest appeared partially dependent on wildtype p53 expression. Mechanistic studies revealed that VPA dramatically enhances HU induced DNA double strand breaks, likely by downregulating the homologous recombination protein, Rad51. The synergistic efficacy of the combination appeared to be maintained in vivo as combination therapy was superior to monotherapies in OCI- AML3 and patient derived xenograft models of AML. Through different selection strategies the work performed in this thesis effectively identified a diverse group of small molecules worthy of further investigation for the treatment of AML. Mechanistic studies provided novel insights into how each of the agents exert their anti-leukemic properties and should guide biomarker development and identification of sensitive patient subgroups. The preclinical animal studies performed in papers II and III provide an important step towards clinical translation.Doktorgradsavhandlin

    Pharmacogenomics of sickle cell disease therapeutics: pain and drug metabolism associated gene variants and hydroxyurea-induced post-transcriptional expression of miRNAs

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    Sickle cell disease (SCD) is a common blood disease caused by a single nucleotide substitution (c.20T>A, p.Glu6Val) in the beta globin gene on chromosome 11. The prevalence of the disease is high throughout large areas in sub-Saharan Africa, the Mediterranean basin, the Middle East, and India due to the level of protection that the sickle cell trait, provides against severe malaria. Approximately 300,000 infants are born per year with sickle cell anemia, which is defined as homozygosity for the sickle hemoglobin (HbS). The majority (nearly 75%) of these births occur in sub-Saharan Africa, particularly in two countries: Nigeria, and the Democratic Republic of the Congo where there are poorly resourced healthcare systems. Early diagnosis, penicillin prophylaxis, blood transfusions, hydroxyurea, and hematopoietic stem-cell transplantation can dramatically improve survival and quality of life for patients with SCD. However, our understanding of the role of genetic and clinical factors in explaining the complex phenotypic diversity of this disease is still limited. Early prediction of the severity, and patients' responses to specific therapeutics of SCD could lead to more precise treatment and management. Beyond well-known modifiers of disease severity, such as fetal hemoglobin (HbF) levels and αthalassemia, other genetic variants might influence specific sub-phenotypes. New treatments and management strategies accounting for these genetic and nongenetic factors could substantially and rapidly improve the quality of life and reduce health care costs for patients with SCD. Patients with SCD are subjected to long term administration of drugs and there is a limited data on pharmacogenomics of SCD therapeutics. Vaso-occlusive crisis (VOC) are the main clinical events of SCD and are associated with recurrent and long-term use of antalgics/opioids and HU. This project aimed to investigate the clinical and genetic predictors of painful vaso-occlusive crisis (VOC) among SCD Cameroon patients by exploring pharmacokinetic determinants of treatment responses as well as post-transcriptional signatures triggered by hydroxyurea treatment, particularly, miRNA expression. SCD patients were recruited from Yaounde Central Hospital and Laquintinie Hospital in Douala (Wonkam et al., 2018, Mnika et al., 2019 (b)), and recent migrants SCD patients from the DRC, recruited at the Haematology Clinic, Groote Schuur Hospital in Cape Town, South Africa (Mnika et al., 2019 (a) and Mnika et al., 2019 (b)). Sociodemographic and clinical data were collected by means of a structured questionnaire. Patients' medical records were reviewed to extract their clinical features over the past 3 years. Specifically, the occurrences of VOC, hematological parameters, hospital outpatient visits, hospitalisation, overt strokes, blood transfusions, and administration of hydroxyurea were recorded. Height, weight, body mass index (BMI), systolic and diastolic blood pressures (SBP and DBP) were measured. Detailed descriptions of patients and sampling methods used in the Cameroonian patients have been reported previously (Wonkam et al., 2018 Mnika et al., 2019 (a) and Mnika et al., 2019 (b)). For the purpose of comparing frequencies of variants, ethnically matched Cameroonian controls were randomly recruited from apparently healthy blood donors in Yaounde for participation in the study. All blood samples were collected for genomic characterisation and analysis. DNA was extracted from peripheral blood, following instructions on the available commercial kit [QIAamp DNA Blood Maxi Kit ® (Qiagen, United States)]. Genotyping (TaqMan and MassArray) was performed for 40 variants in 17 pain-related genes, three fetal haemoglobin (HbF)-promoting loci, two kidney dysfunction-related genes, and HBA1/HBA2 genes for 436 patients. A subset of these samples was also genotyped to analyse 32 core and 267 extended pharmacogenes using commercially available PharmacoScan® platform for characterisation of pharmacokinetic determinant of response. We also compared the pharmacogenes variants from these African groups, to data extracted from the 1000 genomes Project. Moreover, association studies were carried out on pharmacogenes variants with SCD clinical variability. Additionally, protein-protein interaction (PPI) network and enriched biological processes and pathways were investigated. For association studies, statistical models using regression frameworks to analyse 40 variants were performed in R®. For miRNA expression, total RNA was isolated using the miRNeasy kit according to protocol of the Manufacturer (QIAGEN, Hilden, Germany); and sequenced by the Genomic and RNA Profiling Core at Baylor College of Medicine, United States, using the NanoString Platform (NanoString Technologies, Inc., Seattle, WA, United States), according to manufacturer's instructions. Genes with statistically significant changes in expression were analysed using the significance analyses of microarrays (SAM) tools. Female sex, body mass index, Hb/HbF, blood transfusions, leucocytosis and consultation or hospitalisation rates significantly correlated with VOC. Three painrelated gene variants correlated with VOC (CACNA2D3-rs6777055, P = 0·025; DRD2- rs4274224, P = 0·037; KCNS1-rs734784, P= 0·01). Five pain-related gene variants correlated with hospitalization/consultation rates (COMT-rs6269, P = 0·027; FAAHrs4141964, P = 0·003; OPRM1- rs1799971, P = 0·031; ADRB2-rs1042713; P < 0·001; UGT2B7-rs7438135, P = 0·037). The 3·7 kb HBA1/HBA2 deletion correlated with increased VOC (P = 0·002). HbF-promoting loci variants correlated with decreased hospitalisation (BCL11A-rs4671393, P = 0·026; HBS1L-MYB-rs28384513, P = 0·01). APOL1 G1/G2 correlated with increased hospitalisation (P = 0·048). A commercial genotyping array platform (PharmacoScan®) with 4627 markers located in 1191 genes was used to investigate 299 pharmacogenes (32 ADME core and 267 extended pharmacogenes). Based on the PharmacoScan analyses, no statistically significant differences in allele frequencies were detected between SCD cases and controls from Cameroon. A principal component analysis (PCA) revealed that Cameroonians' data clustered with other Africans, but this population is significantly distinct from American, European and Asian populations data. Variant allele frequencies in 21/32 core pharmacogenes were significantly different between the two SCD groups (Cameroon vs. Congo). No correlation between clinical variability and variants in the core genes was detected for both populations under study. An association study of the core and extended PharmacoScan variants to VOC identified statistically significant associations between two single nucleotide polymorphisms (SNPs) to VOC after correction of multiple testing. These two SNPs mapped to 50 genes, with two SNPs located in core pharmacogenes (SLCO4A1- rs118042746, p=1.21e-07; UGT1A10, UGT1A8- rs10176426, p=1.22e-07). Functional enrichment analyses revealed that these 50 genes are involved in three biological processes and four pathways relevant to SCD pathophysiology, including xenobiotic glucuronidation (GO:0052697, p = 2.3e-03), and drug metabolism - other enzymes (p = 2.1e-02). Further analyses of the 50 genes, identified key genes in human proteinprotein networks: NTSR1, LRMDA, SMAD SMAD4 and CDH2. These four genes also interacted with three core pharmacogenes associated with VOC: UGT1A8, UGT1A10 and SLCO4A1. We found 22/798 miRNAs to be differentially expressed under HU treatment, with the majority (13/22) being functionally associated with HbF-regulatory genes, including BCL11A (miR-148b-3p, miR-32-5p, miR-340-5p, miR-29c-3p), MYB (miR-105-5p), KLF-3 (miR-106b-5), and SP1 (miR-29b-3p, miR-625-5p, miR-324-5p, miR-125a-5p, miR-99b-5p, miR-374b-5p, miR-145-5p). The present thesis started by highlighting the scarcity of studies investigating variable responses to pain in SCD patients and then proceeded to addressing this research gap. To our knowledge this is the first body of from Africa to provide evidence supporting the possible development of a genetic risk model for pain in SCD. This is also the first body of work to report an association between these two SNPs and VOC in core and extended pharmacogenes. Our data reveals that the commercial pharmacogenes arrays investigated might need additional evidence for appropriateness among Africans. Therefore, it advocates the need to invest in research exploring population-specific arrays, drug design, targeting, and efficacy, for improved clinical management of patients of African descent. Previous studies have investigated various mechanisms to understand the genomic variations affecting responses to HU, but full understanding of the variable HU-mediated HbF production among individuals affected by SCD remains elusive. The present study showed that mechanisms of HbF production in response to HU, could particularly be mediated through miRNA regulation. The data reveals some alternative perspectives and routes towards identifying new therapeutic targets and approaches for SCD. However, this study needs to be replicated in larger samples in multiple African populations

    EPR Effect-Based Tumor Targeted Nanomedicine

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    I am honored to undertake the work for Guest Editor for this Special Issue of EPR Effect-Based Tumor Targeted Nanomedicine for the Journal of Personalized Medicine. It has already been 35 years since we published the concept of the EPR effect for the first time. The discovery of the new concept of EPR effect gave an impetus effect of growth momentum in nanomedicine, and numerous works are focused on tumor delivery, although the initial idea was based on vascular permeability in infection-induced inflamed tissue, where we discovered bradykinin in the key mediator of vascular permeability.I know, however, there are pros and cons to EPR effect. Cons stem either from a poor understanding of EPR effect, or somehow a biased view of the EPR effect, or from the tumor models being used, particularly in the clinical settings where vascular blood flow is so frequently obstructed. I hope scientists in the clinic, or basic researchers working on the tumor drug delivery, will join the forum of this Special Issue and express their data and opinions.The scope of this issue includes an in-depth understanding of the EPR effect, and issues associated with tumor microenvironment and also further exploitation of EPR effect in human cancer. In addition, new strategies for enhancement of the EPR effect using nanomedicine will be welcome, which is as important as the EPR effect itself. These papers cover not only cancer therapy, but also imaging techniques using nanofluorescent agents, including photodynamic therapy for inflammation, and boron neutron capture therapy

    The Synthesis and Biological Assessment of Novel Ursolic Acid Derivatives as Potential Chemotherapeutic Agents for Cancer

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    Glioblastoma (GBM) is considered to be the most biologically aggressive type of brain tumour accounting for approximately 48% of all malignant primary brain tumours. GBM patients diagnosed have poor prognosis with a low five-year survival rate of The aim of this study was to develop novel UA derivatives to enhance its bioavailability. Nine novel UA derivatives: three different diamine linkers, with Boc-protected and deprotected ends, and with folic acid were designed and synthesized to improve compound activity and/or delivery. The structures of the newly synthesised compounds were confirmed using mass spectrometry, FTIR, 1H NMR and 13C NMR. The structural activity relationship (SAR) of UA and novel UA derivatives that were designed to improve its activity and bioavailability (predicted ADMET profile) were explored, and molecular docking studies against proposed targets – FOLR1 and/or VRK1 were utilised, with an in-depth analysis of predicted interactions. The cytotoxic activity was determined using alamarBlueTM cell viability assay in a panel of cancer cell lines and normal cells. It was found that the conjugation of folic acid to UA decreased its cytotoxic activity. Interestingly, our study showed that the Boc-protected compounds have delayed cytotoxicity in comparison to deprotected compounds, which are more evident in U-251 MG and A431 cell lines using 2D cell culture assay. Whereas in 3D cell culture, only deprotected compounds exhibited an activity. The deprotected novel UA derivatives also retained the inherent anti-proliferative and anti-migratory effect of UA in U-251 MG cells. The synergistic studies focusing on the cell membrane damage performed showed that novel UA derivatives (7 – 12) may have a protective effect when exposed to radioactivity. In addition, the initial inhibitor studies suggests that compounds 8 and 10 at longer timepoint, may trigger multiple cell demise pathway. Based on these preliminary results, this study provides a new insight into using novel UA derivatives and a possible MOA for their anti-cancer effect

    Ultrasensitive detection of toxocara canis excretory-secretory antigens by a nanobody electrochemical magnetosensor assay.

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    peer reviewedHuman Toxocariasis (HT) is a zoonotic disease caused by the migration of the larval stage of the roundworm Toxocara canis in the human host. Despite of being the most cosmopolitan helminthiasis worldwide, its diagnosis is elusive. Currently, the detection of specific immunoglobulins IgG against the Toxocara Excretory-Secretory Antigens (TES), combined with clinical and epidemiological criteria is the only strategy to diagnose HT. Cross-reactivity with other parasites and the inability to distinguish between past and active infections are the main limitations of this approach. Here, we present a sensitive and specific novel strategy to detect and quantify TES, aiming to identify active cases of HT. High specificity is achieved by making use of nanobodies (Nbs), recombinant single variable domain antibodies obtained from camelids, that due to their small molecular size (15kDa) can recognize hidden epitopes not accessible to conventional antibodies. High sensitivity is attained by the design of an electrochemical magnetosensor with an amperometric readout with all components of the assay mixed in one single step. Through this strategy, 10-fold higher sensitivity than a conventional sandwich ELISA was achieved. The assay reached a limit of detection of 2 and15 pg/ml in PBST20 0.05% or serum, spiked with TES, respectively. These limits of detection are sufficient to detect clinically relevant toxocaral infections. Furthermore, our nanobodies showed no cross-reactivity with antigens from Ascaris lumbricoides or Ascaris suum. This is to our knowledge, the most sensitive method to detect and quantify TES so far, and has great potential to significantly improve diagnosis of HT. Moreover, the characteristics of our electrochemical assay are promising for the development of point of care diagnostic systems using nanobodies as a versatile and innovative alternative to antibodies. The next step will be the validation of the assay in clinical and epidemiological contexts

    27th Fungal Genetics Conference

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    Program and abstracts from the 27th Fungal Genetics Conference Asilomar, March 12-17, 2013

    Annual Report

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    Unlocking the Binding and Reaction Mechanism of Hydroxyurea Substrates as Biological Nitric Oxide Donors

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    Hydroxyurea is the only FDA approved treatment of sickle cell disease. It is believed that the primary mechanism of action is associated with the pharmacological elevation of nitric oxide in the blood; however, the exact details of this are still unclear. In the current work, we investigate the atomic level details of this process using a combination of flexible-ligand/flexible-receptor virtual screening coupled with energetic analysis that decomposes interaction energies. Utilizing these methods, we were able to elucidate the previously unknown substrate binding modes of a series of hydroxyurea analogs to hemoglobin and the concomitant structural changes of the enzyme. We identify a backbone carbonyl that forms a hydrogen bond with bound substrates. Our results are consistent with kinetic and electron paramagnetic resonance (EPR) measurements of hydroxyurea–hemoglobin reactions, and a full mechanism is proposed that offers new insights into possibly improving substrate binding and/or reactivity
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