Roles for Structural Biology in the Discovery of Drugs and Agrochemicals Targeting Sterol 14α-Demethylases

Antifungal drugs and antifungal agrochemicals have significant limitations. These include several unintended consequences of their use including the growing importance of intrinsic and acquired resistance. These problems underpin an increasingly urgent need to improve the existing classes of antifungals and to discover novel antifungals. Structural insights into drug targets and their complexes with both substrates and inhibitory ligands increase opportunity for the discovery of more effective antifungals. Implementation of this promise, which requires multiple skill sets, is beginning to yield candidates from discovery programs that could more quickly find their place in the clinic. This review will describe how structural biology is providing information for the improvement and discovery of inhibitors targeting the essential fungal enzyme sterol 14α-demethylase.

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Review on fungal enzyme inhibitors – potential drug targets to manage human fungal infections

RSC Advances ◽

10.1039/c6ra01577h ◽

2016 ◽

Vol 6 (48) ◽

pp. 42387-42401 ◽

Cited By ~ 7

Author(s):

Jayapradha Ramakrishnan ◽

Sudarshan Singh Rathore ◽

Thiagarajan Raman

Keyword(s):

Drug Targets ◽

Enzyme Inhibitors ◽

Fungal Infections ◽

Invasive Fungal Infections ◽

Potential Drug ◽

Fungal Enzyme ◽

Potential Applications ◽

Potential Drug Targets

The potential applications of enzyme inhibitors for the management of invasive fungal infections are explored.

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Platforms for High-Throughput Screening and Force Measurements on Fungi and Oomycetes

Micromachines ◽

10.3390/mi12060639 ◽

2021 ◽

Vol 12 (6) ◽

pp. 639

Author(s):

Yiling Sun ◽

Ayelen Tayagui ◽

Sarah Sale ◽

Debolina Sarkar ◽

Volker Nock ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Drug Targets ◽

Control Strategies ◽

Pathogenic Fungi ◽

Antifungal Drugs ◽

Plant Diseases ◽

Force Measurements ◽

Technical Developments ◽

Alternative Drug

Pathogenic fungi and oomycetes give rise to a significant number of animal and plant diseases. While the spread of these pathogenic microorganisms is increasing globally, emerging resistance to antifungal drugs is making associated diseases more difficult to treat. High-throughput screening (HTS) and new developments in lab-on-a-chip (LOC) platforms promise to aid the discovery of urgently required new control strategies and anti-fungal/oomycete drugs. In this review, we summarize existing HTS and emergent LOC approaches in the context of infection strategies and invasive growth exhibited by these microorganisms. To aid this, we introduce key biological aspects and review existing HTS platforms based on both conventional and LOC techniques. We then provide an in-depth discussion of more specialized LOC platforms for force measurements on hyphae and to study electro- and chemotaxis in spores, approaches which have the potential to aid the discovery of alternative drug targets on future HTS platforms. Finally, we conclude with a brief discussion of the technical developments required to improve the uptake of these platforms into the general laboratory environment.

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Opportunistic yeast pathogen Candida spp.: Secreted and membrane-bound virulence factors

Medical Mycology ◽

10.1093/mmy/myab053 ◽

2021 ◽

Author(s):

Si Jie Lim ◽

Mohd Shukuri Mohamad Ali ◽

Suriana Sabri ◽

Noor Dina Muhd Noor ◽

Abu Bakar Salleh ◽

...

Keyword(s):

Virulence Factors ◽

Drug Targets ◽

Fungal Infections ◽

Antifungal Drugs ◽

Host Immune System ◽

Structural Studies ◽

Candida Spp ◽

Structural Investigations ◽

Membrane Bound ◽

Aspartyl Proteinases

Abstract Candidiasis is a fungal infection caused by Candida spp. especially Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. Although the medicinal therapeutic strategies have rapidly improved, the mortality rate due to candidiasis has continuously increased. The secreted and membrane-bound virulence factors (VFs) are responsible for fungal invasion, damage and translocation through the host enterocytes besides the evasion from host immune system. VFs such as agglutinin-like sequences (Als), heat shock protein 70, phospholipases, secreted aspartyl proteinases (Sap), lipases, enolases and phytases are mostly hydrolases which degrade the enterocyte membrane components except for candidalysin, the VF acts as a peptide toxin to induce necrotic cell lysis. To date, structural studies of the VFs remain underexplored, hindering their functional analyses. Among the VFs, only secreted aspartyl proteinases and agglutinin-like sequences have their structures deposited in Protein Data Bank (PDB). Therefore, this review scrutinizes the mechanisms of these VFs by discussing the VF-deficient studies of several Candida spp. and their abilities to produce these VFs. Nonetheless, their latest reported sequential and structural analyses are discussed to impart a wider perception of the host-pathogen interactions and potential vaccine or antifungal drug targets. This review signifies that more VFs structural investigations and mining in the emerging Candida spp. are required to decipher their pathogenicity and virulence mechanisms compared to the prominent C. albicans. Lay Abstract Candida virulence factors (VFs) including mainly enzymes and proteins play vital roles in breaching the human intestinal barrier and causing deadly candidiasis. Limited VFs’ structural studies hinder deeper comprehension of their mechanisms and thus the design of vaccines and antifungal drugs against fungal infections.

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Posttreatment Antifungal Resistance among Colonizing Candida Isolates in Candidemia Patients: Results from a Systematic Multicenter Study

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01763-15 ◽

2015 ◽

Vol 60 (3) ◽

pp. 1500-1508 ◽

Cited By ~ 28

Author(s):

R. H. Jensen ◽

H. K. Johansen ◽

L. M. Søes ◽

L. E. Lemming ◽

F. S. Rosenvinge ◽

...

Keyword(s):

Genetic Relatedness ◽

Acquired Resistance ◽

Antifungal Drugs ◽

Its Sequencing ◽

Ionization Time ◽

Content Type ◽

Flight Mass Spectrometry ◽

Echinocandin Resistance ◽

Resistance Rates ◽

Initial Blood

The prevalence of intrinsic and acquired resistance among colonizingCandidaisolates from patients after candidemia was investigated systematically in a 1-year nationwide study. Patients were treated at the discretion of the treating physician. Oral swabs were obtained after treatment. Species distributions and MIC data were investigated for blood and posttreatment oral isolates from patients exposed to either azoles or echinocandins for <7 or ≥7 days. Species identification was confirmed using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and internal transcribed spacer (ITS) sequencing, susceptibility was examined by EUCAST EDef 7.2 methodology, echinocandin resistance was examined byFKSsequencing, and genetic relatedness was examined by multilocus sequence typing (MLST). One hundred ninety-three episodes provided 205 blood and 220 oral isolates. MLST analysis demonstrated a genetic relationship for 90% of all paired blood and oral isolates. Patients exposed to azoles for ≥7 days (n= 93) had a significantly larger proportion of species intrinsically less susceptible to azoles (particularlyCandida glabrata) among oral isolates than among initial blood isolates (36.6% versus 12.9%;P< 0.001). A similar shift toward species less susceptible to echinocandins among 85 patients exposed to echinocandins for ≥7 days was not observed (4.8% of oral isolates versus 3.2% of blood isolates;P> 0.5). Acquired resistance inCandida albicanswas rare (<5%). However, acquired resistance to fluconazole (29.4%;P< 0.05) and anidulafungin (21.6%;P< 0.05) was common inC. glabrataisolates from patients exposed to either azoles or echinocandins. Our findings suggest that the colonizing mucosal microbiota may be an unrecognized reservoir of resistantCandidaspecies, especiallyC. glabrata, following treatment for candidemia. The resistance rates were high, raising concern in general for patients exposed to antifungal drugs.

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Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility–mass spectrometry

Biochemical Journal ◽

10.1042/bcj20190318 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3125-3139 ◽

Cited By ~ 2

Author(s):

Daniel Shiu-Hin Chan ◽

Jeannine Hess ◽

Elen Shaw ◽

Christina Spry ◽

Robert Starley ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Structural Biology ◽

Ion Mobility ◽

Biosynthetic Pathway ◽

Screening Tool ◽

Native Mass Spectrometry ◽

Ion Mobility Mass Spectrometry ◽

E Coli ◽

Structural Insights

Abstract CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility–mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

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Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bj20110002 ◽

2011 ◽

Vol 436 (3) ◽

pp. 729-739 ◽

Cited By ~ 23

Author(s):

Marcio V. B. Dias ◽

William C. Snee ◽

Karen M. Bromfield ◽

Richard J. Payne ◽

Satheesh K. Palaninathan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Structural Investigation ◽

Catalytic Mechanism ◽

Shikimate Pathway ◽

Structural Rearrangement ◽

Competitive Inhibitors ◽

Structural Insights ◽

Potential Drug Targets

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19–24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

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Structural biology and structure–function relationships of membrane proteins

Biochemical Society Transactions ◽

10.1042/bst20180269 ◽

2018 ◽

Vol 47 (1) ◽

pp. 47-61 ◽

Cited By ~ 7

Author(s):

Rosana Reis ◽

Isabel Moraes

Keyword(s):

Membrane Proteins ◽

Structure Function ◽

Structural Biology ◽

Drug Targets ◽

Three Dimensional ◽

Data Bank ◽

Technical Advances ◽

Medical Importance ◽

G Protein Coupled ◽

Important Drug

Abstract The study of structure–function relationships of membrane proteins (MPs) has been one of the major goals in the field of structural biology. Many Noble Prizes regarding remarkable accomplishments in MP structure determination and biochemistry have been awarded over the last few decades. Mutations or improper folding of these proteins are associated with numerous serious illnesses. Therefore, as important drug targets, the study of their primary sequence and three-dimensional fold, combined with cell-based assays, provides vital information about their structure–function relationships. Today, this information is vital to drug discovery and medicine. In the last two decades, many have been the technical advances and breakthroughs in the field of MP structural biology that have contributed to an exponential growth in the number of unique MP structures in the Protein Data Bank. Nevertheless, given the medical importance and many unanswered questions, it will never be an excess of MP structures, regardless of the method used. Owing to the extension of the field, in this brief review, we will only focus on structure–function relationships of the three most significant pharmaceutical classes: G protein-coupled receptors, ion channels and transporters.

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Genetic Analysis of Azole Resistance by Transposon Mutagenesis in Saccharomyces cerevisiae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.11.2731 ◽

1999 ◽

Vol 43 (11) ◽

pp. 2731-2735 ◽

Cited By ~ 15

Author(s):

D. P. Kontoyiannis

Keyword(s):

Saccharomyces Cerevisiae ◽

Drug Targets ◽

Molecular Mechanisms ◽

Screening Tool ◽

Transposon Mutagenesis ◽

Antifungal Drugs ◽

Azole Resistance ◽

Recessive Mutations ◽

Novel Drug ◽

Novel Drug Targets

ABSTRACT The increasing resistance of Candida species to fluconazole is cause for concern. To determine the molecular mechanisms involved in resistance to fluconazole, I used a scheme of transposon mutagenesis in Saccharomyces cerevisiae, a genetically tractable yeast that is closely related to Candida albicans. This technique, which permits the generation and analysis of multiple random Tn3::LEU2::lacZfusions, can be used as a disruption mutagen (N. B. Burns et al., Genes Dev. 8:1087–1105, 1994). By using the Tn3::LEU2::lacZlibrary as a disruption mutagen, I found recessive mutations in genes that were previously found to be involved in azole resistance, e.g.,PDR5 and CPR1, and in genes previously found to be involved in azole sensitivity, e.g., ERG3. This approach also enabled me to identify recessive mutations in three genes not previously known to be involved in azole sensitivity. Two of the genes,ADA3 and SPT7, are general transcriptional regulators; the third, YMR034c, is a putative sterol transporter. Finally, by screening the Tn3::LEU2::lacZlibrary for lacZ fusions induced by a low concentration of fluconazole, I identified genes known to be induced by azoles as well as a variety of other genes not previously known to be induced by the drug. In conclusion, transposon mutagenesis is a promising screening tool for use in identifying novel drug targets and in uncovering the mechanisms involved in the response of S. cerevisiae to antifungal drugs.

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Antifungal Resistance, Metabolic Routes as Drug Targets, and New Antifungal Agents: An Overview about Endemic Dimorphic Fungi

Mediators of Inflammation ◽

10.1155/2017/9870679 ◽

2017 ◽

Vol 2017 ◽

pp. 1-16 ◽

Cited By ~ 22

Author(s):

Juliana Alves Parente-Rocha ◽

Alexandre Melo Bailão ◽

André Correa Amaral ◽

Carlos Pelleschi Taborda ◽

Juliano Domiraci Paccez ◽

...

Keyword(s):

Drug Targets ◽

Antifungal Agents ◽

Fungal Infections ◽

Public Health Problem ◽

Antifungal Drugs ◽

Fungal Resistance ◽

Immunocompromised Patients ◽

Dimorphic Fungi ◽

Novel Drug ◽

Novel Drug Targets

Diseases caused by fungi can occur in healthy people, but immunocompromised patients are the major risk group for invasive fungal infections. Cases of fungal resistance and the difficulty of treatment make fungal infections a public health problem. This review explores mechanisms used by fungi to promote fungal resistance, such as the mutation or overexpression of drug targets, efflux and degradation systems, and pleiotropic drug responses. Alternative novel drug targets have been investigated; these include metabolic routes used by fungi during infection, such as trehalose and amino acid metabolism and mitochondrial proteins. An overview of new antifungal agents, including nanostructured antifungals, as well as of repositioning approaches is discussed. Studies focusing on the development of vaccines against antifungal diseases have increased in recent years, as these strategies can be applied in combination with antifungal therapy to prevent posttreatment sequelae. Studies focused on the development of a pan-fungal vaccine and antifungal drugs can improve the treatment of immunocompromised patients and reduce treatment costs.

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Take Me Home, Protein Roads: Structural Insights into Signal Peptide Interactions during ER Translocation

International Journal of Molecular Sciences ◽

10.3390/ijms222111871 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11871

Author(s):

A. Manuel Liaci ◽

Friedrich Förster

Keyword(s):

Structural Biology ◽

Signal Peptide ◽

Target Location ◽

Signal Recognition Particle ◽

Signal Peptidase ◽

Signal Recognition ◽

Signal Sequences ◽

Peptide Interactions ◽

Er Targeting ◽

Structural Insights

Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short peptides, mostly located at the N-termini of proteins, are highly diverse. For most proteins targeted to the ER, it is the interactions between the signal sequences and the various ER targeting and translocation machineries such as the signal recognition particle (SRP), the protein-conducting channel Sec61, and the signal peptidase complex (SPC) that determine the proteins’ target location and provide translocation fidelity. In this review, we follow the signal peptide into the ER and discuss the recent insights that structural biology has provided on the governing principles of those interactions.

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