High-throughput screening for drug discovery targeting the cancer cell-microenvironment interactions in hematological cancers

Introduction The interactions between leukemic blasts and cells within the bone marrow environment affect oncogenesis, cancer stem cell survival, as well as drug resistance in hematological cancers. The importance of this interaction is increasingly being recognized as a potentially important target for future drug discoveries and developments. Recent innovations in the high throughput drug screening-related technologies, novel ex-vivo disease-models, and freely available machine-learning algorithms are advancing the drug discovery process by targeting earlier undruggable proteins, complex pathways, as well as physical interactions (e.g. leukemic cell-bone microenvironment interaction). Area covered In this review, the authors discuss the recent methodological advancements and existing challenges to target specialized hematopoietic niches within the bone marrow during leukemia and suggest how such methods can be used to identify drugs targeting leukemic cell-bone microenvironment interactions. Expert opinion The recent development in cell-cell communication scoring technology and culture conditions can speed up the drug discovery by targeting the cell-microenvironment interaction. However, to accelerate this process, collecting clinical-relevant patient tissues, developing culture model systems, and implementing computational algorithms, especially trained to predict drugs and their combination targeting the cancer cell-bone microenvironment interaction are needed.Peer reviewe

Multiscale Modeling of Tuberculosis Disease and Treatment to Optimize Antibiotic Regimens

Tuberculosis (TB) is one of the world’s deadliest infectious diseases. Caused by the pathogen Mycobacterium tuberculosis (Mtb), the standard regimen for treating TB consists of treatment with multiple antibiotics for at least six months. There are a number of complicating factors that contribute to the need for this long treatment duration and increase the risk of treatment failure. Person-to-person variability in antibiotic absorption and metabolism leads to varying levels of antibiotic plasma concentrations, and consequently lower concentrations at the site of infection. The structure of granulomas, lesions forming in lungs in response to Mtb infection, creates heterogeneous antibiotic distributions that limit antibiotic exposure to Mtb. Microenvironments in the granuloma can shift Mtb to phenotypic states that have higher tolerances to antibiotics. We can use computational modeling to represent and predict how each of these factors impacts antibiotic regimen efficacy and granuloma sterilization. In this thesis, we utilize an agent-based, computational model called GranSim that simulates granuloma formation, function and treatment. We present a method of incorporating sources of heterogeneity and variability in antibiotic pharmacokinetics to simulate treatment. Using GranSim to simulate treatment while accounting for these sources of heterogeneity and variability, we discover that individuals that naturally have low plasma antibiotic concentrations and granulomas with high bacterial burden are at greater risk of failing to sterilize granulomas during antibiotic treatment. Importantly, we find that changes to regimens provide greater improvements in granuloma sterilization times for these individuals. We also present a new pharmacodynamic model that incorporates the synergistic and antagonistic interactions associated with combinations of antibiotics. Using this model, we show that in vivo antibiotic concentrations impact the strength of these interactions, and that accounting for the actual concentrations within granulomas provides greater predictive power to determine the efficacy of a given antibiotic combination. A goal in improving antibiotic treatment for TB is to find regimens that can shorten the time it takes to sterilize granulomas while minimizing the amount of antibiotic required. With the number of potential combinations of antibiotics and dosages, it is prohibitively expensive to exhaustively simulate all combinations to achieve these goals. We present a method of utilizing a surrogate-assisted optimization framework to search for optimal regimens using GranSim and show that this framework is accurate and efficient. Comparing optimal regimens at the granuloma scale shows that there are alternative regimens using the antibiotic combination of isoniazid, rifampin, ethambutol and pyrazinamide that could improve sterilization times for some granulomas in TB treatment. In virtual clinical trials, these alternative regimens do not outperform the regimen of standard doses but could be acceptable alternatives. Focusing on identifying alternative regimens that can improve treatment for high risk patients could help to significantly decrease the global burden for TB. Overall, this thesis presents a computational tool to evaluate antibiotic regimen efficacy while accounting for the complicating factors in TB treatment and improves our ability to predict new regimens that can improve clinical treatment of TB.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/166103/1/cicchese_1.pd

Chemogenomic model identifies synergistic drug combinations robust to the pathogen microenvironment

Antibiotics need to be effective in diverse environments in vivo. However, the pathogen microenvironment can have a significant impact on antibiotic potency. Further, antibiotics are increasingly used in combinations to combat resistance, yet, the effect of microenvironments on drug-combination efficacy is unknown. To exhaustively explore the impact of diverse microenvironments on drug-combinations, here we develop a computational frame-work-Metabolism And GENomics-based Tailoring of Antibiotic regimens (MAGENTA). MAGENTA uses chemogenomic profiles of individual drugs and metabolic perturbations to predict synergistic or antagonistic drug-interactions in different microenvironments. We uncovered antibiotic combinations with robust synergy across nine distinct environments against both E. coli and A. baumannii by searching through 2556 drug-combinations of 72 drugs. MAGENTA also accurately predicted the change in efficacy of bacteriostatic and bactericidal drug-combinations during growth in glycerol media, which we confirmed experimentally in both microbes. Our approach identified genes in glycolysis and glyoxylate pathway as top predictors of synergy and antagonism respectively. Our systems approach enables tailoring of antibiotic therapies based on the pathogen microenvironment

A search for novel synergistic combinations against prevalent fungal phytopathogens

With the global population rising, the emergence of new types or variants of crop pathogens (phytopathogens), and the increasing resistance of these organisms to current crop treatments, there is an urgent need to improve global food security. Each year, fungi destroy crops that could feed up to 600 million people annually. Wheat, the most widely grown crop worldwide, has the second highest yield of all crops, with a global production of 778.5 million metric tonnes in 2021/2022. Zymoseptoria tritici, a major pathogen of wheat, causes Septoria Tritici Blotch (STB). This organism is responsible for around 70% of annual fungicide usage in the EU and up to 20% of annual yield losses in the UK. Novel treatments involving synergistic combinations of inhibitory compounds could improve efficacy while decreasing cost and toxicity to both non-target organisms and the wider environment. In this thesis, novel synergistic combinations of agents, primarily targeting Z. tritici, were sought. The initial focus was on protein translation as a target. Specifically, it was hypothesised that pairs of agents disrupting the fidelity of protein translation could be effective at synergistically inhibiting phytopathogenic fungi. To examine this, rational combinations of compounds were tested against fungi of interest. Several synergies against Botrytis cinerea and Z. tritici were found among compounds of interest. A series of different combinations involving the dual targeting of aminoacylation (attachment of an amino acid to a tRNA), with one compound inhibiting a tRNA synthetase, and a second compound targeting the biosynthesis of the corresponding amino acid, yielded several synergies. One such synergy was between tavaborole, an inhibitor of the leucyl-tRNA synthetase, and chlorimuron ethyl, an inhibitor of the non-cognate branched-chain amino acids. This synergy decreased the minimum inhibitory concentrations (MIC) for each compound by four-fold when in combination, compared to individual application. This proof of principle for dual targeting – inhibiting both biosynthesis and aminoacyl tRNA synthetase function for particular amino acids – opens up the possibility for dozens of potential synergistic combinations targeting the availability of different amino acids for protein translation, and hence growth, of undesirable fungi. For fungi pathogenic to humans, one or both targets should be the availability of essential amino acids to decrease cytotoxicity risks. Furthermore, a powerful synergy against Z. tritici was discovered. This synergy decreased the MICs of the two compounds involved, cyprodinil and diphenyleneiodonium, by 16- and 32-fold when in combination, compared to individual application. However, this synergy was less potent in B. cinerea and Saccharomyces cerevisiae. The mechanism behind this synergy was investigated, initially testing for synergistic mechanisms based around the NADH kinase Pos5p and the electron transport chain, as these were reported as potential targets for the compounds. Experiments suggested that the mechanism most likely involves the synergistic increase of oxidative damage within mitochondria, although some experiments contradict this. Diphenyleneiodonium, an inhibitor of NAD(P)H oxidases, was evidenced to confer increased sensitivity to the pro-oxidant H2O2. The specific action of cyprodinil in the synergy remains unclear while characteristics of increased oxidative stress were seen. Finally, two synergies of interest were tested on live wheat plants to determine if they could protect crops from Z. tritici infection. Experiments showed that at the concentrations used, these synergistic combinations were unable to effectively inhibit the pathogenicity of Z. tritici. Nevertheless, this poor reproduction of laboratory findings in real infections highlights one difficulty with the discovery of novel treatments against fungal phytopathogens, viable for real-world applications. Despite the poor success in preventing Z. tritici infection in the small trial with wheat at the end of this study, the potency of inhibitory-agents acting synergistically, demonstrated in the laboratory here, remains a strategy of high potential. The synergies discovered in this research could be tested against other undesirable fungi with the possibility of successfully controlling infection, and the mechanisms of synergistic action proposed here may provide a foundation to inform future combinatorial testing

Novel approaches for the control of fungal pathogens

Fungal pathogens are a continual threat with potential impacts on human health, agriculture, food and goods security. Despite this, currently used treatments are limited to a handful of drug or fungicide classes. The limited availability of treatment options is further challenged by growing fungal resistance, tightening legislation over drug/fungicide use and evolving public opinion. In this thesis, certain novel approaches were explored for their potential in the control of fungal pathogens of humans or crops. One approach utilised the concept of combinatorial treatments, applied specifically to synergistic interactions among natural product (NP) compounds. NPs have been questioned for their translational applications due to promiscuous activity; this study proposed the potential of synergy for potentiating antifungal activity and improving target specificity. In a high-throughput screening approach, selected NPs were screened pairwise against a wider NP chemical library. Screening of 800 NP combinations revealed 34 pairs that were potentially synergistic in their inhibitory effects on yeast growth. Moreover, scaled-up validation tests for three combinations of particular interest showed that synergy was present against several important pathogens. One synergistic combination was explored mechanistically and found to promote synergistic mitochondrial membrane depolarization and ROS formation. This work indicated the potential for synergistic NP combinations in fungal pathogen control. An additional study focussed on relationships between NP interactions and their underlying mechanisms of synergy, focusing on a particular triangle of NP interactions (involving two synergies but also no interaction). Results indicated that the NP sclareol, found to synergise with a number of other NPs, could also induce synergy between the previously non-synergistic pair of compounds. Results supported that this action of sclareol involved uncoupling of oxidative phosphorylation, which may be an activity that enables synergies against fungal pathogens more widely. An additional approach explored the potential of collateral sensitivity (CS) as a potential drug-repurposing strategy against azole-resistant Candida albicans. CS is where resistance to one drug is linked to sensitivity to another, so offering means to target drug resistant strains. Two azole-resistant clinical isolates of C. albicans showed hypersensitivity to several non-antifungal drugs, particularly aminoglycosides. The mutants were slow growers, but slow growth was not sufficient to explain the hypersensitivity, neither were the isolates’ alleles of erg11, the gene encoding the lanosterol demethylase targeted by azoles. Moreover, the hypersensitivity was not reproduced in other azole-resistant isolates. Mechanistic studies pointed to a possible role for cell wall glycosylation or integrity defects in the original two isolates. Further work expanded the search for CS compounds against azole-resistant C. albicans through a screen of a 1,280-compound library. The results did not identify any hit compounds, but reproducibility and dosage concerns meant that hit compounds could have been missed. A final approach set out to assess mechanistic bases for reported fungal anti-attachment properties of certain polymer materials. One strategy was an accelerated evolution experiment, designed to select C. albicans variants hyper-attaching to polymer. However, attachment propensity did not change, indicating resilience of the anti-attachment material properties. Another strategy examined cell wall properties that may affect anti-attachment, in C. albicans and the plant pathogen Zymoseptoria tritici. Results with selective fluorescent probes highlighted certain cell wall components that were enriched in polymer-attaching or glass-attaching cells. This offers a path for understanding cell properties important for (anti-) attachment to the polymer materials, valuable for informing design of improved polymers. Taken together the three approaches explored in this thesis offer exciting potential for bolstering efforts to control fungal pathogens, providing bases for further mechanistic and possible translational developmen

A search for novel synergistic combinations against prevalent fungal phytopathogens

With the global population rising, the emergence of new types or variants of crop pathogens (phytopathogens), and the increasing resistance of these organisms to current crop treatments, there is an urgent need to improve global food security. Each year, fungi destroy crops that could feed up to 600 million people annually. Wheat, the most widely grown crop worldwide, has the second highest yield of all crops, with a global production of 778.5 million metric tonnes in 2021/2022. Zymoseptoria tritici, a major pathogen of wheat, causes Septoria Tritici Blotch (STB). This organism is responsible for around 70% of annual fungicide usage in the EU and up to 20% of annual yield losses in the UK. Novel treatments involving synergistic combinations of inhibitory compounds could improve efficacy while decreasing cost and toxicity to both non-target organisms and the wider environment. In this thesis, novel synergistic combinations of agents, primarily targeting Z. tritici, were sought. The initial focus was on protein translation as a target. Specifically, it was hypothesised that pairs of agents disrupting the fidelity of protein translation could be effective at synergistically inhibiting phytopathogenic fungi. To examine this, rational combinations of compounds were tested against fungi of interest. Several synergies against Botrytis cinerea and Z. tritici were found among compounds of interest. A series of different combinations involving the dual targeting of aminoacylation (attachment of an amino acid to a tRNA), with one compound inhibiting a tRNA synthetase, and a second compound targeting the biosynthesis of the corresponding amino acid, yielded several synergies. One such synergy was between tavaborole, an inhibitor of the leucyl-tRNA synthetase, and chlorimuron ethyl, an inhibitor of the non-cognate branched-chain amino acids. This synergy decreased the minimum inhibitory concentrations (MIC) for each compound by four-fold when in combination, compared to individual application. This proof of principle for dual targeting – inhibiting both biosynthesis and aminoacyl tRNA synthetase function for particular amino acids – opens up the possibility for dozens of potential synergistic combinations targeting the availability of different amino acids for protein translation, and hence growth, of undesirable fungi. For fungi pathogenic to humans, one or both targets should be the availability of essential amino acids to decrease cytotoxicity risks. Furthermore, a powerful synergy against Z. tritici was discovered. This synergy decreased the MICs of the two compounds involved, cyprodinil and diphenyleneiodonium, by 16- and 32-fold when in combination, compared to individual application. However, this synergy was less potent in B. cinerea and Saccharomyces cerevisiae. The mechanism behind this synergy was investigated, initially testing for synergistic mechanisms based around the NADH kinase Pos5p and the electron transport chain, as these were reported as potential targets for the compounds. Experiments suggested that the mechanism most likely involves the synergistic increase of oxidative damage within mitochondria, although some experiments contradict this. Diphenyleneiodonium, an inhibitor of NAD(P)H oxidases, was evidenced to confer increased sensitivity to the pro-oxidant H2O2. The specific action of cyprodinil in the synergy remains unclear while characteristics of increased oxidative stress were seen. Finally, two synergies of interest were tested on live wheat plants to determine if they could protect crops from Z. tritici infection. Experiments showed that at the concentrations used, these synergistic combinations were unable to effectively inhibit the pathogenicity of Z. tritici. Nevertheless, this poor reproduction of laboratory findings in real infections highlights one difficulty with the discovery of novel treatments against fungal phytopathogens, viable for real-world applications. Despite the poor success in preventing Z. tritici infection in the small trial with wheat at the end of this study, the potency of inhibitory-agents acting synergistically, demonstrated in the laboratory here, remains a strategy of high potential. The synergies discovered in this research could be tested against other undesirable fungi with the possibility of successfully controlling infection, and the mechanisms of synergistic action proposed here may provide a foundation to inform future combinatorial testing

Novel approaches for the control of fungal pathogens

Fungal pathogens are a continual threat with potential impacts on human health, agriculture, food and goods security. Despite this, currently used treatments are limited to a handful of drug or fungicide classes. The limited availability of treatment options is further challenged by growing fungal resistance, tightening legislation over drug/fungicide use and evolving public opinion. In this thesis, certain novel approaches were explored for their potential in the control of fungal pathogens of humans or crops. One approach utilised the concept of combinatorial treatments, applied specifically to synergistic interactions among natural product (NP) compounds. NPs have been questioned for their translational applications due to promiscuous activity; this study proposed the potential of synergy for potentiating antifungal activity and improving target specificity. In a high-throughput screening approach, selected NPs were screened pairwise against a wider NP chemical library. Screening of 800 NP combinations revealed 34 pairs that were potentially synergistic in their inhibitory effects on yeast growth. Moreover, scaled-up validation tests for three combinations of particular interest showed that synergy was present against several important pathogens. One synergistic combination was explored mechanistically and found to promote synergistic mitochondrial membrane depolarization and ROS formation. This work indicated the potential for synergistic NP combinations in fungal pathogen control. An additional study focussed on relationships between NP interactions and their underlying mechanisms of synergy, focusing on a particular triangle of NP interactions (involving two synergies but also no interaction). Results indicated that the NP sclareol, found to synergise with a number of other NPs, could also induce synergy between the previously non-synergistic pair of compounds. Results supported that this action of sclareol involved uncoupling of oxidative phosphorylation, which may be an activity that enables synergies against fungal pathogens more widely. An additional approach explored the potential of collateral sensitivity (CS) as a potential drug-repurposing strategy against azole-resistant Candida albicans. CS is where resistance to one drug is linked to sensitivity to another, so offering means to target drug resistant strains. Two azole-resistant clinical isolates of C. albicans showed hypersensitivity to several non-antifungal drugs, particularly aminoglycosides. The mutants were slow growers, but slow growth was not sufficient to explain the hypersensitivity, neither were the isolates’ alleles of erg11, the gene encoding the lanosterol demethylase targeted by azoles. Moreover, the hypersensitivity was not reproduced in other azole-resistant isolates. Mechanistic studies pointed to a possible role for cell wall glycosylation or integrity defects in the original two isolates. Further work expanded the search for CS compounds against azole-resistant C. albicans through a screen of a 1,280-compound library. The results did not identify any hit compounds, but reproducibility and dosage concerns meant that hit compounds could have been missed. A final approach set out to assess mechanistic bases for reported fungal anti-attachment properties of certain polymer materials. One strategy was an accelerated evolution experiment, designed to select C. albicans variants hyper-attaching to polymer. However, attachment propensity did not change, indicating resilience of the anti-attachment material properties. Another strategy examined cell wall properties that may affect anti-attachment, in C. albicans and the plant pathogen Zymoseptoria tritici. Results with selective fluorescent probes highlighted certain cell wall components that were enriched in polymer-attaching or glass-attaching cells. This offers a path for understanding cell properties important for (anti-) attachment to the polymer materials, valuable for informing design of improved polymers. Taken together the three approaches explored in this thesis offer exciting potential for bolstering efforts to control fungal pathogens, providing bases for further mechanistic and possible translational developmen