2.

HIT TO LEAD AND LEAD OPTIMIZATION

• The ultimate goal of hit-to-lead and lead optimization is to identify candidate drugs with the
best possible balance of potency, target selectivity, pharmacokinetics and safety, to give drug
development programs the maximum chance of success in the clinical development phase.
• High-throughput screening (HTS) is done which identifies potential drug candidates (hits), and
the next step is the hit-to-lead process, where researchers strive to refine and improve upon the
initial hits, to generate more potent and selective lead compounds.
• The hit-to-lead and lead optimization process require robust biological testing, medicinal
chemistry and rigorous safety assessments in order to refine and improve the chemical
structures and biological activity of the compounds.
• A hit compound is a molecule that exhibits activity at the target of interest
• A hit is a compound identified during the early stages of drug discovery that shows activity
against a specific biological target, such as a protein or receptor, in a screening assay.
• Hits are typically compounds that show activity against the target in a primary screen
• A lead compound is a candidate drug whose structure has been chemically optimized and
whose activity has been rigorously tested via biological assays.
• During HTS, millions of compounds may be screened, typically identifying hundreds to
thousands of hits.
• The hit-to-lead and lead optimization process refines this number down to one or two candidate
molecules for clinical development.
• The hit-to-lead stage refines initial hits to identify lead compounds that possess improved drug-
like characteristics.
• HTS harnesses automation and miniaturization to screen libraries of thousands to millions of
compounds against the drug target in parallel
• Hits are discovered via techniques like high-throughput screening (HTS), fragment-based drug
design (FBDD), or virtual screening (analyzing large chemical libraries in silico (using
computer simulations

Steps involved in the Hit-to-Lead Process

1. Hit Identification
• Hits demonstrate activity against a biological target, often with weak potency.
• Hits are identified through high-throughput screening (HTS) eg fragment-based drug
discovery, virtual screening of natural product libraries
• Initial hits normally lack drug-like properties and require optimization
2. Hit Validation
• Hits are re-tested to confirm activity – elimination of false positives
• Confirm activity through secondary assays.
 • Structure-activity relationship (SAR) studies begin to evaluate specificity for the target
and rule out false positives

3. Lead Optimization
• Hits are chemically modified to enhance their potency, selectivity, and drug-like properties.
• The target parameters include:
o Potency: Increase target-binding strength (e.g., lower IC₅₀ values).
o Pharmacokinetics: Improve Absorption, Distribution, Metabolism and Excretion
(ADME) properties.
o Toxicity: Reduce off-target effects and potential toxicity.

4.Selection of Leads

• Leads should have good oral bioavailability, selectivity, and stability

• The best candidates chosen based on improved pharmacokinetic and pharmacodynamic
properties
• Toxicity and off-target effects assessed

Lead Optimization

• It is the process of optimizing initial "hit" compounds to improve their drug-like properties,
making them suitable for further development as potential drug leads
• Lead compounds can be either natural or chemical products possessing biological activity
against drug targets.
• Lead optimization is the final phase of drug discovery that focuses on optimizing different
characteristics of lead compounds eg target selectivity, biological activity, potency, and
toxicity potential
• The goal is to develop a candidate with optimal efficacy, safety and drug-like characteristics
suitable for preclinical and clinical development
• To further approve the molecule as a preclinical candidate, the absorption, distribution,
metabolism, excretion, and toxicity (ADMET) properties of the compound are evaluated.
• Animal models are used to analyze the effectiveness of the lead compound in modulating the
disease.
• High-throughput techniques are used in modification of compounds for the purpose of
improved characteristics eg magnetic resonance and mass spectrometry or computational
methods.
• Some of the computational methods used in this phase are pharmacophore studies,
molecular dynamics, QSAR, and molecular docking
• The process involves;
 a. Chemical Modifications: Chemists systematically modify the structure of hit compounds
to determine which parts are essential for activity. Eg Replacing specific functional
groups with analogs or introducing halogen atoms to improve potency or metabolic
stability.
b. Structure-Activity Relationship (SAR): SAR studies map how changes in molecular
structure impact biological activity. This helps to understand which structural features
are most important for efficacy.
c. In Silico Screening: Computational techniques such as molecular docking help
prioritize modifications. Eg compounds can be evaluated for how well they fit into the
active site of a protein target. Molecular docking is used to predict how a small molecule
(ligand) binds to a target protein ( eg enzyme or receptor)
d. ADMET Profiling: Preliminary evaluation of the compound's drug-likeness:
i. Solubility: if the compound dissolve sufficiently in physiological
conditions?
ii. Permeability: Will the compound cross biological membranes (e.g., the
blood-brain barrier)?
iii. Stability: Is the compound stable under physiological conditions?

Aim of lead Optimization

a. Enhance Potency- Maximize the compound's activity against the biological target.
b. Improve Selectivity-Minimize off-target interactions to reduce side effects.
c. Optimize Pharmacokinetics: Achieve favorable absorption, distribution, metabolism, and
excretion (ADME) properties.
d. Address Toxicity- Identify and mitigate potential toxic effects.
e. Enhance Drug-Like Properties: Improve solubility, stability, permeability, and other
physicochemical parameters.
f. Simplify Synthesis-Ensure that the compound can be produced efficiently and cost-effectively

Examples of Hit-to-Lead Successes

• Imatinib (Gleevec)- Initial hits targeting the BCR-ABL kinase in chronic myeloid leukemia
(CML) were refined to improve kinase inhibition. Chemical modifications enhanced
selectivity and reduced off-target effects, leading to imatinib’s development as a breakthrough
therapy.
• Sitagliptin (Januvia): Inhibitors of DPP-4 were identified as hits for managing type 2
diabetes. Through H2L, sitagliptin was optimized to achieve high potency and excellent
pharmacokinetic properties, with minimal off-target activity
• Anti-Malarial Drugs (Artemisinin Derivatives): The hit compound is Artemisinin, a natural
product, demonstrated activity against malaria but had poor pharmacokinetics. A lead
Artemether and artesunate were developed by modifying artemisinin to enhance solubility
and efficacy.
• Kinase Inhibitors for Cancer: The hit was a small molecule inhibitors of kinases (e.g., ATP-
competitive compounds) identified through HTS. A Lead, Imatinib was optimized for selective
inhibition of the BCR-ABL kinase in chronic myeloid leukemia
• HIV Protease Inhibitors: The hit was a weak protease inhibitors identified through
structure-based design. The lead: Saquinavir was developed by optimizing interactions with
the active site of HIV protease

Methods used in H2L and Lead Optimization

i.) Structure-Activity Relationship (SAR) Studies: Systematic modification of the lead structure
to determine how changes affect activity and other properties.
ii.) Structure-Based Drug Design: Use of target protein structures (e.g., from X-ray
crystallography or cryo-EM) to design better-fitting molecules.
iii.) Computational Methods: Virtual screening, molecular dynamics, and docking studies.
The process of predicting the biological activity of large compound collections is known as
virtual screening
Computational methods can also be used to elucidate the energies associated with chemical
reactivity and predict how to improve a synthetic protocol.
iv.) In Vivo Testing: Preclinical animal studies to evaluate efficacy, pharmacokinetics, and
toxicity.

Techniques used in Hit-to-Lead and Lead Optimization

o Structure-Based Drug Design (SBDD)

• Eg X-ray crystallography is used to visualize a compound bound to a target protein.
• The technique has been used in the development of HIV protease inhibitors (e.g., ritonavir)
relied heavily on SBDD to enhance binding affinity.
• Nuclear Magnetic Resonance (NMR) Spectroscopy technique is used to study the structure,
dynamics, and interactions of molecules
o Fragment-Based Drug Discovery (FBDD)
• Fragments with weak binding affinity are optimized by adding functional groups or
linking fragments.
• Eg the development of venetoclax, a BCL-2 inhibitor for chronic lymphocytic leukemia.
• AI Artificial Intelligence tools predict the impact of chemical modifications on potency,
selectivity, and pharmacokinetics.