VSpipe, an Integrated Resource for Virtual Screening and Hit Selection: Applications to Protein Tyrosine Phospahatase Inhibition

et al. 2020

mBio

Self Cite

There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose to human health. Here, we have performed a comprehensive characterization and validation of the promising target methionine synthase (MetH). We show that in Aspergillus fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. Interestingly, growth can be recovered in the presence of certain metabolites, which shows that metH is a conditionally essential gene and consequently should be targeted in established infections for a more comprehensive validation. Accordingly, we have validated the use of the tetOFF genetic model in fungal research and improved its performance in vivo to achieve initial validation of targets in models of established infection. We show that repression of metH in growing hyphae halts growth in vitro, which translates into a beneficial effect when targeting established infections using this model in vivo. Finally, a structure-based virtual screening of methionine synthases reveals key differences between the human and fungal structures and unravels features in the fungal enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is a valuable target for the development of new antifungals. IMPORTANCE Fungal pathogens are responsible for millions of life-threatening infections on an annual basis worldwide. The current repertoire of antifungal drugs is very limited and, worryingly, resistance has emerged and already become a serious threat to our capacity to treat fungal diseases. The first step to develop new drugs is often to identify molecular targets in the pathogen whose inhibition during infection can prevent its growth. However, the current models are not suitable to validate targets in established infections. Here, we have characterized the promising antifungal target methionine synthase in great detail, using the prominent fungal pathogen Aspergillus fumigatus as a model. We have uncovered the underlying reason for its essentiality and confirmed its druggability. Furthermore, we have optimized the use of a genetic system to show a beneficial effect of targeting methionine synthase in established infections. Therefore, we believe that antifungal drugs to target methionine synthase should be pursued and additionally, we provide a model that permits gaining information about the validity of antifungal targets in established infections.

Section: Methodsmentioning

confidence: 99%

Targeting Methionine Synthase in a Fungal Pathogen Causes a Metabolic Imbalance That Impacts Cell Energetics, Growth, and Virulence

Scott

Sueiro-Olivares

et al. 2020

mBio

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“…Our approach to assess the druggability of the AfPPases was based on a combination of virtual screening with VSpipe [15] and pocket analysis with PockDrug [17] in order to identify suitable druggable sites for drug development on the essential AfPPases. Currently, there are no available 3D structures of any of these AfPPases, thus, we had to generate molecular homology models for the virtual screening.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously demonstrated that the virtual screening tool VSpipe could be used to identify functional ligand binding sites in the human phosphatase PTP1B, as well as to guide the selection of initial hits for drug discovery [15]. The blind docking option in VSpipe is useful when there is no previous knowledge about the functional sites on the target protein, as is the case for the AfPPases.…”

Section: Resultsmentioning

confidence: 99%

“…In this study, we used a combination of computational and experimental approaches to expand the ontology-based classification and previous identification of AfPPases [10,14], and we validated three additional phosphatases as essential for viability. Finally, we used a virtual screening pipeline, VSpipe v1.0 [15] to define putative functional and druggable sites in the essential phosphatases. Results from the virtual screening suggest that different modes of inhibition may be possible to block phosphatase function and guide the selection of suitable starting points for drug development.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Identification of Functional and Druggable Sites in Aspergillus fumigatus Essential Phosphatases by Virtual Screening

Johns

Al-Shidhani

et al. 2019

IJMS

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Fungal diseases are a serious health burden worldwide with drug resistance compromising efficacy of the limited arsenal of antifungals available. New drugs with novel mechanisms of action are desperately needed to overcome current challenges. The screening of the Aspergillus fumigatus genome identified 35 phosphatases, four of which were previously reported as essential for viability. In addition, we validated another three essential phosphatases. Phosphatases control critical events in fungi from cell wall integrity to cell cycle, thus they are attractive targets for drug development. We used VSpipe v1.0, a virtual screening pipeline, to evaluate the druggability of the seven essential phosphatases and identify starting points for drug discovery. Targeted virtual screening and evaluation of the ligand efficiency plots created by VSpipe, enabled us to define the most favourable chemical space for drug development and suggested different modes of inhibition for each phosphatase. Interestingly, the identified ligand binding sites match with functional sites (active site and protein interaction sites) reported for other yeast and human homologues. Thus, the VSpipe virtual screening approach identified both druggable and functional sites in these essential phosphatases for further experimental validation and antifungal drug development.

“…This sequence together with the structure of the C. albicans orthologue (CaMetH) (PDB ID: 4L65, DOI: 10.1016/j.jmb.2014.02.006) were used to create the molecular homology model in Modeller (version 9.23) (106) with the basic option mode. The AfMetH model was then used for virtual screening with the semi-automated pipeline VSpipe (107). For comparison we also performed virtual screening with the structure of the human methionine synthase (hMS) containing the folate and homocysteine binding domains (PDB ID: 4CCZ).…”

Section: Molecular Homology Models and Virtual Screeningmentioning

confidence: 99%

Targeting methionine synthase in a fungal pathogen causes a metabolic imbalance that impacts cell energetics, growth and virulence

Scott

Sueiro-Olivares

et al. 2020

Preprint

Self Cite

29There is an urgent need to develop novel antifungals to tackle the threat fungal pathogens pose 30 to human health. In this work, we have performed a comprehensive characterisation and 31 validation of the promising target methionine synthase (MetH). We uncover that in Aspergillus 32 fumigatus the absence of this enzymatic activity triggers a metabolic imbalance that causes a 33 reduction in intracellular ATP, which prevents fungal growth even in the presence of methionine. 34Interestingly, growth can be recovered in the presence of certain metabolites, which evidences 35 that conditional essentiality, defined as genes whose deficiency can be overcome in specific 36 conditions, is present in pathogenic fungi. As this concept must be considered for correct target 37 validation, we have optimised a genetic model to mimic treatment of established infections 38 using the tetOFF system. We show that repression of metH in growing hyphae halts growth in 39 vitro, which translates into a beneficial effect when targeting established infections using this 40 model in vivo. Finally, a structural-based virtual screening of methionine synthases reveals key 41 differences between the human and fungal structures and unravels features in the fungal 42 enzyme that can guide the design of novel specific inhibitors. Therefore, methionine synthase is 43 a valuable target for the development of new antifungals. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59Fungal pathogens represent an increasing risk to human health 1 , with over one billion people 60 worldwide affected by mycoses annually. Many of these mycoses are superficial infections of 61 the skin, nails or mucosal membranes and although troublesome are usually not life-62 threatening. However, some fungi cause devastating chronic and invasive fungal infections, 63 which result in an estimated 1.6 million deaths per year 2 . Incidences of invasive infections 64 caused by Aspergillus, Candida, Cryptococcus and Pneumocystis species are increasing 3 , a cause 65 for serious concern as these genera are responsible for 90% of deaths caused by mycoses 4 . 66Despite the availability of antifungal drugs, mortality rates for invasive aspergillosis, invasive 67 candidiasis, cryptococcal meningitis and Pneumocystis jirovecii pneumonia are intolerably high, 68reaching over 80%, 40%, 50% and 30% respectively 2,5 . There are currently only four classes of 69 antifungals in clinical use to treat invasive infections (azoles, echinocandins, polyenes and 70 flucytosine), all suffering from pharmacological drawbacks including toxicity, drug-drug 71 interactions and poor bioavailability 6,7 . With the sole exemption of flucytosine, which is only 72 used in combinatory therapy with amphotericin B for cryptococcal meningitis and Candida 73 endocarditis 7 , the current antifungals target critical components of the fungal cell membrane 74 or cell wall 8 , which represents a very limited druggable space. The rise of antifungal resistance 75 presents an additional challenge as mortality rates in pat...