Drug Discovery and Development Stages

Drug Discovery Stages

• Modern drug discovery, design, and development occur in a sequential order

Steps in Drug Discovery and Design

• Choose a disease and drug target
• Identify a bioassay, which can be in vitro or in vivo
• Find a "lead compound"
• Identify structure-activity relationship (SARs)
• Identify the pharmacophore
• Improve pharmacodynamics, focusing on drug-target interactions
• Improve pharmacokinetic properties

Development Steps

• Patent the drug
• Study drug metabolism and test for toxicity
• Design a manufacturing process and carry out clinical trials
• Register and market the drug
• Make money

Bioassays

• Choosing the right bioassay or test system is crucial for a successful drug research program
• Tests should be simple, quick, and relevant
• In-vitro tests are preferred because they are cheaper, easier to set up, simple to run, yield results quickly, permit replication, and can be automated
• In-vitro, meaning "within the glass," refers to performing a procedure in a controlled environment outside of a living organism

In-Vitro Tests

• Screens for pharmacological activity
• Enzyme inhibitors can be tested on the pure enzyme in solution
• HIV protease enzyme, cloned and expressed in E. coli, can undergo experiments to determine if an enzyme inhibitor is active or non-active and to determine IC50 values
• Receptor agonists and antagonists can be tested on isolated tissues or cells that express the target receptor on their surface
• The affinity of drugs for receptors, indicating how strongly they bind, can be measured by radioligand studies
• Antibacterial drugs can be tested by measuring how effectively they inhibit or kill bacterial cells in culture

In-Vitro Toxicity Screening

• Cytotoxicity and target organ toxicity can be screened
• Potential hepatotoxic, nephrotoxic, and neurotoxic effects can be investigated in simple in-vitro systems
• Reliable in-vitro tests for teratogenicity, which is the ability to cause defects in a developing fetus, are a priority
• Whole embryo cultures from rats, mice, chicks, fish, and frogs can be used

Benefits of In-Vitro Teratogenicity Assays

• Can considerably reduce the expense and duration of toxicological studies
• Reduces the numbers of animals used

Limitations of In-Vitro Methods

• In-vitro test systems are simplified models of in-vivo processes
• Direct extrapolation of in-vitro data to the in-vivo situation has limitations in both pharmacological and toxicological investigations

Factors Affecting In-Vitro Test Validity

• The target may be artefactual in receptor binding assays or toxicological screens
• Artefactual results are due to the static nature of in-vitro systems and the use of inappropriate drug concentrations
• Compound biokinetics differ in-vitro and in-vivo
• Biotransformation affects pharmacological or toxicological activities, absorption, distribution, and excretion in-vivo

Drug Activity and Solubility

• Drug activity in-vitro relates to its aqueous solubility
• Modifying the most active, polar compound in-vitro to develop more lipophilic congeners is necessary for subsequent oral dosing in in-vivo studies
• Such modifications can alter the drug's pharmacological and toxicological properties
• This is due to effects on absorption, distribution, metabolism, excretion, and kinetics

Concentration Considerations

• The concentrations which drugs are pharmacologically and toxicologically active need consideration
• Lead compounds should be pharmacologically active in-vitro at relevant concentrations
• These concentrations should be likely to be achieved in-vivo with realistic dose levels
• Na+K+-ATPase inhibition with an antidepressant compound at millimolar concentrations is irrelevant, as the compounds are active at submicromolar concentrations

In-Vivo Tests

• In-vivo tests on animals often involve inducing a clinical condition in the animal to produce observable symptoms
• Transgenic animals are often used in-vivo

Uses of Transgenic Animals

• Mouse genes can be replaced with human genes to produce human receptors or enzymes for in-vivo testing
• Mouse genes can be altered to make the animal susceptible to a particular disease (animal model of the disease, e.g., breast cancer) so drugs can be tested to see how well they prevent that disease

Transgenic Animal Definition

• An animal whose genome has been altered by the transfer of a gene or genes from another species or breed using recombinant DNA technology

Problems Associated with In-Vivo Testing

• It is slow and causes animal suffering
• Pharmacokinetic problems- obtained results may be misleading and difficult to rationalize
• Tests might turn out to be invalid
• Observed symptoms might be caused by a different physiological mechanism
• Many antiulcer drugs that proved effective in animal testing were ineffective in humans
• In-vivo testing is still crucial in identifying problems that might be associated with drug use and which cannot be picked up by in-vitro tests

Case study: Drug Discovery Project Using In-Vitro Bioassay

• The project aims to discover Dengue-2 NS2B-NS3 protease inhibitors as potential dengue therapeutics

Dengue Fever Research

• Choose Dengue fever as the disease
• Target system: Dengue virus
• Target molecule: Dengue virus protease
• Identify an in-vitro bioassay
• Find a lead
• Optimisation

Dengue Virus Life Cycle

• The life cycle starts with the attachment and binding of virions on the host cell surface
• This is followed by entry into the cell via endocytosis to form an endosome
• The endosome undergoes fusion, releasing RNA into the cytoplasm
• Viral RNA is translated into a polyprotein, which is cleaved into viral proteins by protease enzyme
• Replication of RNA and virus assembly occur, followed by budding where new mature virions are released

The role of Flaviviruses

• Requires correct processing of their polyprotein by the viral NS2B-NS3 protease
• NS2B-NS3 protease is a potential target for therapeutics against the dengue virus
• The study’s aim is to obtain potential inhibitors towards DEN-2 NS2B-NS3 protease

Bioassay Development

• The target molecule is Dengue virus protease
• Buy the required enzyme or produce the enzyme
• Understand how to cleave the protein (DV protease)
• A bioassay/test method is required to evaluate inhibitory activities of the compounds (inhibitors) towards DV protease

Reversible Inhibitor Interactions

• Competitive inhibition
• Non-competitive inhibition
• Uncompetitive inhibition

Materials for In-Vitro Bioassay

• DV protease enzyme (NS2B-NS3 protease) and a protein as the substrate
• A measurable parameter to assess activity of the enzyme
• Instruments to measure the parameter

NS2B-NS3 Protease Production

• Protein expression and harvesting done using recombinant DNA technology
• Culture E-Coli strain with DEN-2 NS2B-NS3 protease gene
• Protein extraction, purification, verification and quantification
• Followed by Protease activity determination

Purchasing and Using the Substrate

• Use a fluorogenic substrate which is t-Butyloxycarbonylglycyl-L-arginyl-L-arginine-4-metylcoumaryl-7-amide (BOC-Gly-Arg-Arg-MCA)
• Protease activity is determined towards the BOC-Gly-Arg-Arg-MCA substrate
• Proteases cleave the substrate and release the fluorogenic moiety (7-amino-4-methyl coumarin), (AMC)

Fluorometer

• A fluorometer measures the activity of the enzyme
• Fluorescence is used to measure enzyme activity
• Fluorescence intensity of the released AMC can be measured with a ModulusTM II Mocroplate Multimode Reader with excitation at 365 nm and emission at 410-460 nm

Formulas for Concentration

• y represents "Fluorescence Intensity” and x is "AMC Concentration"
• “AMC Concentration” = “Fluorescence Intensity"/ 492.54

Computer Analysis

• Analyzing compounds from NADI, NCI and Purchasable-DBname databases for active hits

Inhibition Assay Conditions

• Reaction mixture has a volume of 100 ul
• Buffer of 200mM Tris-HCl with a pH of 8.5
• D-2 NS2B-NS3 Protease is at the concentration of 0.5uM
• Substrate Boc-GRR-MCA is at the concentration of 100uM
• Incubation period is 60 seconds at 37° C

Sample Preparation

• Sample is dissolved in DMSO and performs
• Perform serial dilutions in 1.5ml tubes using DMSO and ensures
• Total DMSO in reaction mixture remains at 2%

In-Vitro Results

• All compounds are active
• S4 > PA > S1 > S3 > S2
• There is overall no correlation between fit value and Ki activity