Hit-to-lead is a process in drug discovery where a potential drug ‘hit’ or lead compound is identified from a large pool of chemical compounds or natural product extracts that have shown activity against a specific target. The goal of hit-to-lead optimization is to identify a lead compound that has sufficient potency, selectivity and drug-like properties. This process requires a multidisciplinar approach and often starts with high-throughput screening techniques.


  • High throughput screening using robotized system (cell based and phenotypic assays)
  • Gene expression profiling
  • Gene manipulation
  • In vitro target validation
  • In vivo target validation
  • Access to biobank samples

Molecular Biophysics

Protein-protein, solute-solute, solute-lipid interactions

  • Absorbance and fluorescence spectroscopy:  time-correlated single-photon counting
  • Fluorescence anisotropy (steady-state and time-resolved)  surface plasmon resonance
  • Chromatography
  • Fret-melting
  • UV spectroscopy
  • Isothermal titration calorimetry (ITC)
  • HTRF Homogeneous Time Resolved Fluorescence (Cisbio)
  • AlphaScreen- Amplified Luminescence Proximity Homogeneous Assay
  • Biophysical tools to evaluate antibody affinity, stability and potency
  • Fiber-optic SPR (Fox Biosystems)
  • Microfluidic Diffusional Sizing (Fluidic Analytics)

Molecular structure

  • Circular dichroism spectroscopy
  • Fourier-transform infrared spectroscopy
  • Nuclear Magnetic Resonance (NMR)


Aggregation dynamics

  • Dynamic light scattering
  • Absorbance and fluorescence spectroscopy
  • Transmission Electron Microscopy
  • Nanoparticle Tracking Analyser
  • Techniques to evaluate protein stability in forced aggregation assays (MDS; FO-SPR)


  • Cellular and tissue biophysics

Cellular morphology and biomechanics

  • Atomic force microscopy
  • Transmission electron microscopy


Cellular membrane penetration

  • Fluorescence spectroscopy
  • Flow cytometry
  • Confocal microscopy


Computational methods

  • Algorithms and machine learning
  • Molecular docking
  • Molecular dynamics simulations

Phage Display: used to screen large libraries of candidate molecules that act on/interact with a specific target. A gene encoding a protein/peptide of interest is inserted into a phage coat protein gene, causing the phage to “display” the protein on its outside. These displaying phages can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein/peptide and those other molecules. The in vivo phage display consists in the selection of phage libraries using biopannings in living animals. The phages circulate in the organism allowing the expressed molecules at the phage surface to bind directly to the specific target, organ or tissues.

  • Screening of hit (target) binders (leads)
  • Selection of tissue specific leads (peptides/antibodies)
  • Selection of target specific moieties
  • Construction of phage display libraries
  • Optimization of protocols
  • Isolation and identification of targeting phage displayed binders
  • Enrichment screening for specific targets through several rounds
  • Identification of organ specific moieties
  • Next generation sequencing (NGS) of the selected ligands/leads

In vitro Blood Brain Barrier (BBB) permeability evaluation

This assay is often used to screen hit compounds for the treatment of neurological diseases, of CNS tumors and brain metastasis, and of infectious diseases (e.g. SARS-CoV-2, HIV, etc). It is used to evaluate drug candidate’s transport across the BBB and their effect on BBB integrity; to evaluate cytotoxicity and morphological alterations of BBB endothelial cells; to study drug transport mechanisms and to identify potential drug off-target effects in the brain.

In vitro models

  • Human (HBEC-5i) or mouse (bEnd.3) BBB models using confluent cell monolayers cultured in permeable tissue culture inserts (monoculture or co-culture with brain vascular pericytes)


  • Evaluation of markers of BBB integrity: Lucifer Yellow, FD4 or FD40; Transepithelial Electrical Resistance (TEER) readings
  • Determination of compounds concentration: fluorescence intensity measurement or reversed phase HPLC
  • Determination of Apparent Permeability (Papp) of test compounds in both directions

In vitro Intestinal permeability evaluation 

The study of the transport of drug candidates and other molecules across the intestinal epithelium is essential to predict their bioavailability in humans. Caco-2 (human colon adenocarcinoma cell line) cell culture model is widely used to that end as it closely resembles the intestinal epithelium in terms of morphology, function and expression of transport enzymes involved in drug absorption and metabolism. Thus, it can be used to predict oral drug absorption in humans, as well as to evaluate drug-drug interactions, toxicity and pharmacokinetics.


  • Transepithelial Electrical Resistance (TEER) readings
  • Determination of compounds concentration through HPLC
  • Determination of Apparent Permeability (Papp) of test compounds in both directions

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