Team:IISER TVM/project implementation

IGEM-IISERTVM

Project


Implementation

INTRODUCTION

Invasive fungal infections represent a major global health threat in today's world [1][2]. Existing therapeutics not only foreshadow the emergence of drug-resistant fungal strains, but may also have significant cytotoxic and nephrotoxic effects [3]. Our efforts culminated in the development of "Moldemort," a novel antifungal agent, which we believe holds the potential to reduce the global burden of invasive fungal infections significantly.


DRUG ADMINISTRATION AND DELIVERY MECHANISM


In order to achieve precise spatio-temporal and dosage regulation of our chitinases, the team chose nanoencapsulation based on Poly Lactic co-Glycolic Acid (PLGA) Nanoparticles (NP) as a drug delivery system. Being an easily tunable and FDA-approved polymer, PLGA has been widely used for controlled delivery of proteins [4]. Adjustments in the polymer molecular weight , lactide-glycolide ratio and the concentration of the drug will enable us to stably release the desired dosage levels of the chitinase in the desired intervals of time. Our PLGA-NP will be tagged on its surface with Dectin-1 , a mammalian innate immune receptor, which will help our chimeric chitinases bind to beta-glucans present in the fungal cell wall [5]. At the site of infection by the fungi, PLGA will be degraded through ester bond hydrolysis to release our enzymes. Our tissue cells recognize PLGA-NP’s as foreign bodies and accelerate the hydrolysis through enzymatic and free radical reactions [6].


PLGA-NP oligomers have been reported to be extensively soluble in blood [7], and hence intravenous drug delivery is suitable as the primary mode of Moldemort administration to combat the wide variety of fungal infections which spread through the bloodstream e.g. candidiasis. Intravenous administration also is known to prevent degradation of the drug by proteolytic enzymes [8]. Rapid onset of drug action and complete bioavailability of the drug even when administered in low amounts are key advantages of Intravenous Drug Delivery Systems [9].


However, due to the extremely expansive range of infection sites by fungal pathogens, we simultaneously propose alternative delivery routes to help tackle the gamut of fungal infections. For the large number of fungal infections affecting the lungs, the pulmonary route offers immediate absorption due to high surface area and extensive vascularisation [10]. NPs are known to facilitate sustained release of the drug in the lungs followed by circulation through the bloodstream, and hence help in reducing the drug dosage frequency [11]. Odorranalectin coated PLGA-NPs are capable of crossing the blood brain barrier, thereby targeting fungal infections nested in the brain such as Cerebral Aspergillosis [12]. Oral and Transdermal drug delivery systems of PLGA-NPs are made possible by including the necessary modifications to prevent degradation [13] [14].


Our idea benefits from its modularity, both at the levels of enzyme design and drug delivery. Depending on the type of fungal infection, domains from suitable wild-type chitinases can be joined to improve activity towards chitin substrates as well as modify temperature and pH dependent activities of the parent molecule. This flexibility is excellently complemented by the diverse array of PLGA nanoparticles, which as discussed earlier can facilitate regulated enzyme release to various regions of the body. Our solution is thus widely applicable and can be customised based on the class of fungal infection, providing a high level of specificity.


PRE-CLINICAL TESTING:

INVITRO TESTING AND INCELLULO ASSAYS:

Since Moldemort is a group of novel antifungal enzymes intended for therapeutic use in human patients, comprehensive characterisation of their structural and biochemical properties, assessing enzyme activity against diverse fungal species in culture, and utilising in-cellulo and other models (such as organoid, organ-on-chip etc.) of fungal infection to validate the antifungal nature of our enzymes are all essential. We propose the following tests to achieve the previously stated goals:


  1. Circular Dichroism (CD) Spectroscopy:

    This is a spectroscopic technique based on differential absorption of circularly polarised light. In Biology, it is primarily used to analyse the different secondary structural types in proteins or polypeptides such as alpha helices, parallel and antiparallel beta-sheets, turns etc. Hence, we will use CD to elucidate the various secondary structures in our novel enzymes, which will in parallel serve as a quality control mechanism, providing evidence towards proper folding and adequate structural integrity. For this, 200 µL of 3 µM protein solution will be used to assess the presence of secondary structures such as alpha helices and beta sheets by measuring the ellipticity at various wavelengths primarily around 208 nm and 222 nm at 20 °C. In addition, the integrity of the determined secondary structures will be measured by progressively increasing the temperature from 20 °C to 100 °C in intervals of 5 °C, allowing us to generate reliable structural profiles of our recombinant enzymes.


  2. Enzymatic Assays Involving Colloidal Chitin:

    These assays serve as methods to measure the activity of our chitinases using reducing end group N-acetamido-glucose produced from colloidal chitin as a proxy for chitinase activity. A reaction will be set up consisting of 100 μL chitinase enzyme solution and 100 μL of 1%(w/v) colloidal chitin (pH = 5.4) and incubated at 40 °C for 10 minutes. The reaction will be terminated by adding 200 μL 3,5-Dinitrosalicylic acid (DNS reagent) and heating in a boiling water bath for 10 minutes. The colored solution will be centrifuged at 15,000 rotations per minute (rpm) for 5 minutes. The supernatant can then be subjected to spectrophotometric measurement at 540 nm to assess the amount of colloidal chitin which has been degraded. One unit of chitinase activity is defined as the amount of enzyme that liberates 1 µg of N-acetamido-glucose per minute at pH 5.4 and 50 °C [15]. In an alternative protocol, a 2 mL reaction containing 0.5 mL 0.5% colloidal chitin in phosphate buffer (pH = 5.5), 0.5 mL crude enzyme extract, and 1 mL distilled water will be vortexed and incubated in a water bath shaker at 50 °C for 60 minutes. The reaction will be arrested by the addition of 3 mL DNS reagent followed by heating at 100 ◦C for 10 minutes with 40% Rochelle’s salt solution, and the absorption of the appropriately diluted test sample will be measured at 530 nm along with substrate and enzyme blanks. In this case, one unit of enzymatic activity is defined as the amount of enzyme that is required to release 1 mol of N-acetyl-d-glucosamine per minute from 0.5% of dry colloidal chitin solution under assay conditions.


  3. Dependence of chitinase activity on temperature and pH:

    A thorough and precise understanding of the temperature and pH characteristics of our chitinase enzymes is absolutely essential before their use in clinical trials. Ideally, we are looking for the enzymes to retain a significant fraction of their maximum activities at physiological temperatures and pH. For this, we will carry out the following tests:


    • pH Dependence:

      To assess the pH dependence of our enzymes, the purified enzyme/crude extract will be incubated at 37 °C (defined temperature) for 10 minutes at various pH values in the range of 5-11. The buffers used for maintaining pH will be as follows:

      1. 25 mM Sodium acetate (pH 5, 6)
      2. 25 mM Tris HCl (pH 7, 7.5 , 8. 8.5)
      3. 25 mM Carbonate buffer (pH 9, 10 , 11)

      The amount of substrate degraded can be quantified using spectrophotometric measurements as described previously.


    • Temperature dependence:

      After obtaining the optimum pH, the effects of varying temperatures on enzymatic activity will be investigated at the optimum pH. The enzymatic activity will be measured at temperatures from 20 °C to 70 °C, using a protocol similar to the one followed for the pH dependence assay, and a graph of enzymatic activity versus temperature will be plotted.


      The optimum temperature for chitinase activity will be determined by incubating a reaction mixture of enzyme and substrate for 30 minutes at different temperatures in the range 20 °C - 70 °C at pH 7.5.


    • Thermostability of the chitinase:

      Another key characteristic of our enzymes that needs to be thoroughly characterised is their thermostability. In order to assess this, crude enzyme solutions or the purified protein will be incubated without substrate for 3 hours at various temperatures (40 °C, 50 °C, 60 °C, and 70 °C). The activity of the enzyme against chitin substrate will then be assessed at the optimum temperature for enzyme activity as determined prior.


    • Activation energy of the chitinase:

      The chitinase activation energy (Ea ) for chitin hydrolysis will be determined by measuring the reaction rate constant (K) at different temperatures(10 °C - 70 °C), and calculating log K from the Arrhenius equation:



      After plotting log K vs 1/T, the activation energy will be estimated by measuring the slope of the linear portion of the graph.



    • Cylindrical plate assay for antifungal activity:

      This assay relies on fungal colonies grown in Potato Dextrose Agar (PDA) media (composed of potato infusion, dextrose, and agar). Fungal mycelium of the desired species are first inoculated in petri plates containing PDA media. When the colony size reaches 3-4 cm, various wells are made and filled with different concentrations of the pure protein/crude enzyme extract or blank controls. The plates are further incubated at 23 °C until mycelium growth envelops the control wells, and crescents of inhibition around wells with antifungal proteins are produced [15]. Our approach will be similar to the one followed by Kirubakaran and colleagues [15].



      Fig. Inhibitory activity of a plant chitinase towards P. theae and R. solani, shown on the left and right respectively.


    • Observing antifungal activity using Microscopy:

      The aim of this test is to observe deformation of mycelia in the zone of inhibition from the cylindrical plate assay described above. In essence, fungal hyphae from the periphery of the zone of inhibition (ZOI, region around the well in which fungal growth is inhibited) are scrapped, stained with lactophenol blue, and examined under a light microscope to discern any morphological changes. A similar procedure may be followed for a control fungal culture and the observations are compared.


      In addition to the proposed tests, cellular models can also be used as a precursor to animal trials. Firstly, the cytotoxicity of the chitinases will be assessed as described previously [3]. Briefly, A549 (human lung adenocarcinoma) and MRC5 (lung fibroblast) cells will be cultured in Dulbecco’s Modified eagle medium (DMEM; Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (FBS) and 50 μg/mL Penicillin-Streptomycin (PS) antibiotic cocktail, and incubated at 37 °C and 5% CO2. For the experiment, cells will be seeded in a 96 well plate with approximately 5 × 104 cells per well, and 24 hours post plating, treated with various concentrations of our recombinant enzymes ranging from 0.96 to 500 μg/mL as described earlier [3]. Cell viability may be measured by determining the absorbance at 570 - 600 nm using the indicator resazurin at a concentration of 0.01% [3]. In addition, the effectiveness of our chitinases may be tested in a myriad of experimental models, such as cell culture systems, organoids, organ-on-chip, organoid-on-chip, and multi-organ-chip [16], which encapsulate the complexity of living systems in a far more accurate manner than in vitro studies.


ANIMAL TESTING:

After the completion of characterization of the protein, along with in-vitro and in-cellular studies, toxicity testing using an appropriate animal system needs to be conducted.


Carrying out single-dose toxicity using two different rodent species (mice and rats) is essential. In addition to intravenous administration of the drug, alternative routes such as oral drug administration, inhalation, and transdermal drug delivery will be explored, along with testing for any effects of local toxicity. The animals will be observed for 14 days after the administration; the Minimum Lethal Dose (MLD) and Maximum Tolerated Dose (MTD) must be determined. Signs of intoxication, effects on body weight, and gross pathological changes have to be reported.


Repeated dose toxicity testing involves using two mammalian species (one rodent and one non-rodent). The selection of species will be dependent on the comparison between humans and the selected species in terms of pharmacokinetics, metabolic profile, physiology and the expression of the invasive fungal species. The drug will be administered daily for a week using the route intended for delivery. Four grades of doses will be used - a control, low (produces no observable toxicity), intermediate (causes symptoms, but not gross toxicity or death), and the highest dose (causing observable toxicity). Parameters to be monitored include behavioural, physiological, biochemical, and microscopic observations. This dose-ranging study will be followed up with 14-, 28-, 90- and 180- day repeated dose toxicity studies.


In addition to the single and repeated dose toxicity testing, male and female fertility testing, allergenicity, and carcinogenicity testing will be carried out [17].

CLINICAL TRIALS:

Clinical trials in India are divided into 4 phases.

  1. Phase 1- Human Pharmacology studies
  2. Phase 2- Therapeutic Exploratory trials
  3. Phase 3- Therapeutic Confirmatory trials
  4. Phase 4- Post-marketing trials

PHASE 1:

The objective of studies in this phase will be the estimation of safety and tolerability with the initial administration of our drug into humans. Studies in this phase of development usually have non-therapeutic objectives and can be conducted in healthy subjects. Studies done in this phase will help to understand the maximum tolerated dose, pharmacokinetics, and pharmacodynamics of our drug. Maximum tolerated dose studies will help to determine the nature of adverse reactions and the tolerability of the dose range. These studies include both single and multiple-dose administration. Pharmacokinetics studies will help in the characterization of a drug's absorption, distribution, metabolism, and excretion. Pharmacodynamic study results of healthy volunteer subjects in this phase will help in determining the dosage and dose regimen to be applied in later studies.


PHASE 2:

The main objective of this phase of clinical trials will be to evaluate the effectiveness of the new drug in patients with the condition under study and to determine the common short-term side effects and risks associated with the drug. Studies in this phase will be conducted in a group of patients that will be a relatively homogenous population based on some relatively narrow criteria. Doses used in this phase will be usually less than the highest doses used in Phase 1. An important goal of this phase of clinical trials will be to determine the dose and regimen for phase 3 clinical trials.


PHASE 3:

The primary objective of this phase of clinical trials will be the confirmation of therapeutic benefits. The studies in this phase are intended to provide an adequate basis for marketing approval. The studies in this phase will be designed to confirm the results of phase 2 trials that our drug is safe and effective for use in the intended indication and recipient population. The dose-response relationships (relationships among dose, drug concentration in blood, and clinical response), use of the drug in wider populations, in different stages of the disease, or the safety and efficacy of the drug in combination with other drugs will also be studied in this phase.


PHASE 4:

This phase of the post-marketing trial comes after the drug approval. Post marketing surveillance is undertaken to obtain additional information about the risks and benefits resulting from the long-term usage of drugs. The correlation of adverse events reported during this phase with data of animal toxicity studies will help to draw markers for future warnings of potential adverse events likely to occur with other drugs. The studies in this phase will include mortality/morbidity studies, dose-response studies, and clinical trials in a patient population not adequately studied in the pre-marketing phase [18] [19].


IMPLEMENTATION CHALLENGES:

CHALLENGES IN ADMINISTRATION:

Invasive fungal infections have widely varied target organs, ranging from the lungs, sinuses, bloodstream, gastrointestinal tract, and skin, to name a few. Thus, it becomes quite challenging to determine an optimal mode of administration of the chitinase depending upon the target organ.The possibilities of having an adverse immune reaction due to our chimeric chitinase must be explored before the administration of the drug. Animal models will partially help us answer such questions.


ANTIFUNGAL RESISTANCE:

The development of drug resistance is usually an inevitable effect in most pathogenic infections, and fungal diseases are no exception to this. In the case of fungal infections, the course of treatment is often very long, leading to a high probability of drug-resistant fungi being formed. Fungi employ several strategies including dormant spore formation and wall remodelling, significantly altering the composition of the fungal cell wall, potentially reducing the percentage of chitin present, thus helping to escape the action of the drug [20] [21] [22].


COST OF MANUFACTURING:

The estimated cost of our drug-taking into consideration formulation, manufacturing, processing, distribution, and marketing, has to be determined, and a cap on the pricing must be ensured to facilitate the fair distribution of the drug among the various economic classes of the society.


APPLICATIONS IN OTHER AREAS:

DIAGNOSTIC TOOL:

Fluorescently labeled chitinase can be used as a diagnostic tool for invasive fungal diseases, eliminating the time and hassle associated with culturing and direct microscopic examination of the sample. Attaching a fluorescent marker to the chitinase and allowing it to bind to the chitin components of the fungal cell wall, would allow for efficient and effective methods of diagnostics, resulting in better contrast in imaging as well as faster results when compared to the traditional methods [23].


FUNGICIDAL FORMULATION:

Another avenue for the application of our product is as a fungicidal formulation. Damp walls and wood are often subjected to extensive colonization by fungal species. This in addition to being unsightly can also have a deteriorating effect on human health. Exposure to these contaminants has been shown to be associated with the aggravation of several respiratory conditions, and hence removal of this fungal contamination with our chitinases would certainly be beneficial for society. Alternatively, they can also be used as fungicides in a more conventional sense to tackle the variety of fungal infestations that affect the agricultural industry. Traditional methods of control involve using harsh chemicals which have a deleterious effect on the environment as well as our health. On the other hand by modifying and using the formulation of Moldemort we believe that we have a dynamic antifungal agent in our hands, which can be used to get rid of several fungal species safely and efficiently.


CONCLUSION:

Fungal contamination is a nearly ubiquitous issue worldwide due to the ease of transmission and hardy nature of fungal spores. Several species of fungi contaminate walls and other surfaces, damage equipment in factories and laboratories, infect agricultural crops, and cause diseases in humans and other livestock. Thus, they present a significant burden to society and lead to billions of dollars in losses worldwide each year, spread across various sectors. Conventionally used fungicides and antifungal drugs are often limited in their applicability and may have environmental as well as cytotoxic effects. We have developed a class of recombinant chitinases, which we collectively call Moldemort, as a therapeutic agent to tackle the menace of invasive fungal infections. These enzymes combine functional domains from various naturally occuring chitinases, essentially amplifying their antifungal activity. We propose the use of PLGA nanoparticles as a delivery system for our chitinases due to their modularity, customizability, and ability to target drugs to diverse sites in the body, paramount in the fight against fungal infections. We also believe that due to the essential nature of chitin for fungi, the probability of development of drug resistant strains is low. Along with their inherent therapeutic value, our chitinases may find applications in diverse sectors such as diagnosis of fungal infections and as a fungicide for agriculture. Thus, we strongly believe in the power of Moldemort to have a positive impact when it comes to reducing the burden of fungi on society!


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