Antifungal compounds in development against
Invasive Candidiasis (IC) presents a global mortality rate greater than 40%, occupying the fourth place worldwide as the most frequent opportunistic nosocomial disease. Although the genus Candida consists of around 200 species, only 20 are reported as etiological agents of IC, being Candida albicans the most frequent causal agent. Even when there is a broad range of antifungals drugs for Candida infections, azoles, polyenes, and echinocandins are considered among the most effective treatment. However, there is some incidence for antifungal resistance among some Candida strains, limiting treatment options. Several molecular mechanisms with antifungal agents have been reported for C. albicans where insertions, deletions, and point mutations in genes codifying target proteins are frequently related to the antifungal drug resistance. Furthermore, gene overexpression is also frequently associated to antifungal resistance as well as an increase in the activity of proteins that reduce oxidative damage. This chapter summarizes the main molecular mechanisms to C. albicans antifungal drug resistance, besides offering an overview of new antifungal agents and new antifungal targets to combat fungal infections.
- resistance mechanism
Nowadays, the most widely used antifungal drugs for IC include: A) azoles, for instance fluconazole (FLZ), itraconazole (ITC), voriconazole, posaconazole, isavuconazole; B) polyenes such as amphotericin B (AMB); C) echinocandins like caspofungin, micafungin, and anidulafungin [9, 10, 11].
These antifungal compounds act on different parts of the fungal cell (Figure 1). Azoles interrupt the ergosterol biosynthesis, the main component of the fungal membranes [10, 12, 13]. Polyenes such as AMB interact with ergosterol making pores in the cell membrane [10, 12, 13, 14]; while echinocandins act blocking the synthesis of β-d-glucan located in the fungal cell wall [13, 15]. The gradual risk increment for Candida infection and the greater use of antifungal agents has increased resistance towards
The following chapter offers an overview of the main genetic mechanisms contributing to the antifungal resistance in
2. Molecular mechanisms of antifungal resistance
Fungi cell membrane is mainly integrated by ergosterol, a sterol contributing to several cellular functions, besides modulating membrane fluidity and the structure and function of membrane proteins. The azoles mechanism of action is to inhibit 14α-lanosterol demethylase, encoded by the
The evolution of antimicrobial agent’s resistance is common, as there are many microbes able to develop strategies against drugs action. The incremented azoles resistance is mainly a result of their fungistatic rather than fungicidal nature [17, 18, 19]. The mechanisms of resistance to azole antifungal agents have been elucidated in
2.1.1 Mutations of the ERG11 target enzyme
Mutations in the
Some clinical isolates share common mutations with environmental azole-resistant strains, suggesting that some azole-resistant clinical isolates could have their origin in the environment . This resistance appears to be driven by the agricultural use of azoles. In patients without azoles treatment, resistance has been identified derived from the environment. These cases involved a Cyp51A substitution at position 98 (from leucine to histidine), and a 34 base tandem repeat (TR) in the cyp51A promoter, leading to overexpression. Both changes are necessary to confer resistance. In particular, these resistant isolates can be crossed with susceptible strains, suggesting that resistance could be transferred through the sexual cycle. Strains with these alterations have emerged throughout Europe and beyond. Additionally, a new environmentally selected resistance mutation (TR46, Y121F, T289A) was reported among patients in the Netherlands .
2.1.2 Dysregulation of the target enzyme ERG11
One way to decrease the drug effective concentration is the overexpression of
2.1.3 Alteration of the ergosterol biosynthesis pathway (point mutations in ERG genes)
Brief exposures of two to three hours to azoles cause transient upregulation of the
Modification of the metabolic pathway can be effective at different points, as example, alteration of the last steps of biosynthesis through the inactivation of the
Four clinically isolated
2.1.4 Efflux pumps
A mechanism to decrease the azoles intracellular concentration is increasing their output. This class of resistance is mediated by the activity of transport systems such as the pleiotropic drug resistance (PDR) class of ATP-binding cassette transporters (ABC) and major facilitators superfamily (MFS) transporters . These membrane proteins translocate compounds across cell membranes actively using different energy sources. ABC proteins are primary transporters that use ATP hydrolysis. MFS pumps are secondary transporters that use the motive force of the proton across the plasma membrane. Both types of transporters contain distinctive protein domains that confer substrate specificity: nucleotide-binding domains (NBD) in ABC pumps and transmembrane domains (TMD) in ABC and MFS pumps. Fungal PDR proteins appear to share common features on both sides of the two TMDs that separate the cytosolic from the outer cytosolic space [18, 26]. This probably reflects the fact that the cytosolic part is the motor that drives the transport of a variety of substrates through the lipid bilayer through the core of the protein into the outer cytosolic space or the outer layer of the lipid bilayer .
The up-regulation of ABC and MFS transporters is mediated by specific regulations in resistant fungal pathogens. Point mutations defined as gain-of-function (GOF) mutations in these regulators confer an inherently high level of expression of the transporters in drug-resistant strains. GOF mutations in the transcription factor Upc2p led to increased resistance to fluconazole in
The potent fungicidal activity of polyenes derives from their ability to selectively bind sterol at the fungal cell membrane (Figure 1). Four models have been proposed as the mode of action for polyenes: 1) the pore formation model, 2) the surface adsorption model, 3) the sterol sponge model, and 4) the oxidative damage model . The pore formation model is the most studied mechanism, where polyenes are directly intercalated with the ergosterol membrane forming ion channels that permeabilize and kill yeast cells [14, 27]. Additionally, indirect mechanisms of fungal cells damage have been identified due to the effect of polyene compounds, such as those mediated by reactive oxygen species (ROS) and by the secretion of interleukin-1β (IL-1β) by host cells [28, 29].
The polyene AMB is a broad-spectrum drug and is one of the main antifungals used for ICs [10, 14]. AMB is heptane isolated from
2.2.1 Alteration in the composition of sterols in the cell membrane (mutations in
The most common mechanism for acquired resistance to AMB in
2.2.2 Response to oxidative stress and alterations in the cell wall
Fungal resistance mechanisms are also related to oxidative stress regulation, allowing the cell to tolerate exposure to AMB [14, 30]. In
Echinocandins are lipo-peptides that inhibit 1,3-β-d-glucan synthetase, which is responsible for the biosynthesis of 1,3-β-d-glucan, one of the main components of the fungal cell wall, causing osmotic instability and therefore the death of fungal cells (Figure 1) [10, 13]. This class of drugs has certain advantages attributable to its effects on the fungal cell wall, including a lower risk of side effects since animal cells do not have this structure . Echinocandins have a limited spectrum, but for
Echinocandins are the first major new class of antifungal drugs on the market in decades. Consequently, it is of vital importance to assess the nature of the resistance mechanism to this class of drugs. Mutations that affect the target site are the most likely resistance mechanism that exists (Figure 2), since unlike azoles, echinocandins are poor substrates for drug exit through efflux transporters, ruling out this mechanism of resistance [10, 13]. Specific mutations have already been reported in two highly conserved regions of the Fks1 subunit of glucan synthetase, a membrane protein, which can confer resistance
2.3.1 Acquired FKS mutations
Resistance-associated amino acid substitutions occur in two highly conserved hot-spot (HS) regions of the FKS genes. The residues they encompass are Phe641– Pro649 and Arg1361 in
2.3.2 Adaptive stress responses
The fungal cell wall is a dynamic structure that changes during growth and development, requires 1,3-β-d-glucan crosslinking, an essential polymer for the survival of the fungal cell. Echinocandins alter the integrity of the cell wall and induce stress in the cell. In response to this, the fungal cell possesses a repertoire of mechanisms to protect the cell against such destabilization. Protection against cell wall weakening is induced through a variety of stress adaptation mechanisms, which involve protein kinase C (PKC), calcineurin, and Hsp90 [10, 13]. Stress signals in the cell wall are transmitted through the Rho GTPase, which mobilizes various effectors. Its activation alters several carbohydrate polymers along with the structure and remodelling of the cell wall. The Hsp90 heat shock protein organizes a cellular stress response circuit that has a major impact on resistance to echinocandins. Also, the genetic or chemical modulation of the Hsp90 protein reduces tolerance to echinocandins . In response to the inhibition of FKS by the action of echinocandins, a greater amount of chitin is produced helping to maintain the integrity of the cell wall as chitin replaces 1,3-β-d-glucan, thus reducing sensitivity to drugs [10, 13, 48].
3. New antifungals
The resistance of
3.1 Discovery and development of new antifungal drugs
This part of the chapter provides an overview of ongoing efforts to develop new classes of antifungal drugs (Table 1). Although there are several strategies for the development of these drugs, these include those obtained from new chemical agents, from reusing existing drugs, from peptides with antimicrobial properties, and finally from natural compounds extracted from plants [10, 55, 58].
|Source||Compound||Target||Mechanism of action||Reference|
|Rezafungin (CD101)||β-d-glucan||β-d-glucan synthase inhibition|||
|Ibrexafungerp (SCY-078)||β-d-glucan||β-glucan synthase inhibition|||
|VT-1161||Ergosterol||Specific for fungal Cyp51|||
|Fosmanogepix (APX001]||Glycosyl phosphatidylinositol||GPI biosynthesis inhibition|||
|Aureobasidin A||Inositol phosphorylceramide synthase||Sphingolipids biosynthesis inhibition|||
|Efungumab (or Mycograb)||HSP90||Antibody binds to fungal HSP90|||
|Geldanamycin-like agents||HSP90||HSP90 inhibition|||
|AR-12||Probably blocks fungal acetyl-CoA synthetase 1||Downregulation of chaperone proteins|||
|T − 2307||Mitochondrial membrane potential||Respiratory chain complexes inhibition|||
|VL-2397 (ASP2397)||Unknown||Unknown, but taken up by Sitl|||
|Rifampin||RNA polymerase||Enhance the antifungal activity|||
|Verapamil||Calcium channel||Enhance the antifungal activity|||
|Lysozyme||Secreted aspartic protease (SAP)||Reduces SAP activity and secretion|||
|Lactoferrin (hl.f)||Antimicrobial activity||Production of cationic antimicrobial peptide lactoferricin|||
|Human b-defensins (HBD)||Cell membrane||Increases membrane permeability|||
|Histatin-5||Non-lytic ATP efflux||Inhibition of adhesion|||
|Cathelicidins||Cell membrane||Increases membrane permeability|||
|Unknown||Induces apoptosis in |||
|Unknown||Inhibits hyphal growth in |||
|Germination||Inhibits the formation of germination tube and biofilms in |||
|Thymol (terpene)||Ergosterol||Binds to ergosterol in the membrane resulting in cell death|||
|Carvacrol (terpene)||Cell membrane||Alters cellular cytoplasmic membrane and induces apoptosis|||
Several new chemical-antifungals are designed specifically to target either 1,3-β-d-glucan (such as Rezafungin and Ibrexafungerp) or ergosterol (such as the compound VT-1161). These compounds are very specific for fungal infections or they have a longer half-life, offering better efficacy [58, 60, 61, 62]. At the same time, several of these antifungal agents have new targets and subsequently, new mechanisms of action. For instance, fosmanogepix, formerly APX001, and aureobasidin A, which act by inhibiting inositol acyltransferase, and inositol phosphorylceramide synthase, respectively [63, 64]. Efungumab (or Mycograb) and geldanamycin-like agents can inhibit the HSP90 chaperone, which has been also shown to confer resistance to antifungals [65, 66]. The AR-12 compound deregulates chaperone’s activity by blocking fungal acetyl-CoA synthase . The T-2307 compound is an arylamidine that inhibits the respiratory chain complex and is active against yeast and filamentous fungi . Finally, the VL-2397 compound has a similar structure to the ferrichrome siderophore, and whose mechanism of action or its target is unknown, but it is known to be transported by the Sit1 protein . Some compounds that have been already tested for other types of diseases are now receiving a new focus as antifungals. These include two compounds that enhance the antifungal activity, such as rifampin, which acts on RNA polymerase , and verapamil, which acts on a calcium channel . We have also given importance to alternative compounds such as peptides and plant extracts; many molecules are actually studied with promising results, especially against
3.2 New targets and alternative approaches
Despite the efforts made to discover, repositioning, or create new antifungal drugs, it is imperative to find new targets to help eliminating
Finally, an alternative approach to conventional antifungal drugs is the use of nanotechnology, which produces the so-called “nanoantibiotics”. These nanoantibiotics are unique due to their improved physicochemical properties, such as reduced toxicity and biocompatibility as well as their size that must be less than 100 nm . The antimicrobial properties of silver have been known for a long time, so silver nanoparticles were tested as antimicrobials and showed potent activity against drug-resistant fungal biofilms .
A better understanding of the resistance mechanisms of azoles, polyenes, and echinocandins, along with the discovery of new cellular and clinical factors promoting resistance, will facilitate the design of more effective strategies to overcome and prevent resistance to antifungal agents. Even though several biomedical research offer a window hoping to reduce the incidence of
ALMR thanks the National Council of Science and Technology of Mexico (CONACyT) for the postdoctoral fellowship granted. RIAS and KCP are thankful for the scholarship granted by the National Council of Science and Technology of Mexico (CONACyT).
Conflict of interest
All authors declare no conflicting interests.