Methods to determine drug target residence time and to select best drug-target candidates

A method to determine the residence time between at least one Target and at least one Ligand, said Target contained in a complex cellular mixture of at least one of further Targets and other biomolecules, comprising the following steps:

• Step A:
• Incubating said at least one Target with said at least one Ligand, for incubation period of time with a binding reaction between said Ligand and said Target,
• Step B:
• Stopping said binding reaction by removing said at least one Ligand in as far as not bound to Target;
• or reducing the number of binding events by reducing the concentration of the Ligand,
• Step C:
• Incubating said at least one Ligand bound to said at least one Target for different periods of time, leading to an incubated Ligand bound mixture,
• Step D:
• Limited proteolysis of the incubated Ligand bound mixture under a condition in which said at least one Target is in the original conformational state to be detected leading to a first fragment sample,
• wherein this step D is followed by either the sequence of:
• Step E:
• Denaturation of the sample to a denaturated first fragment sample,
• Step F. Digestion of the sample to obtain a completely fragmented sample, or this Step D is followed by the following step:
• Step G:
• Removal of large peptides and proteins or other biomolecules from said first fragment sample to form an enriched fragment sample,
• for both cases followed by the steps:
• an analytical analysis, wherein quantitative mass spectrometry-based assays in the form of at least one of selected reaction monitoring parallel reaction monitoring, and data-independent acquisition of product ion spectra is used,
• wherein for the case of Steps E and F, the analytical analysis is run on the completely fragmented sample for the determination of fragments characteristic being the result of both the limited proteolysis step D. as well as of the complete fragmentation step F,
• and wherein for the case of Step G the analytical analysis is run on the enriched fragment sample,
• measurement of peptide abundance over distinct incubation times post Ligand removal, to determine the dissociation half-life,
• the residence time (τ) being calculated therefrom according to the following equation
• τ
• =
• 1
• koff
• =
• t
• ⁢
• 1
• /
• 2
• ln
• ⁢
• 2
• =
• t
• ⁢
• 1
• /
• 2
• 0.693
• (
• Equation
• ⁢
• 1
• )
• with
• τ
• =
• Residence
• ⁢
• time
• k
• off
• =
• dissociation
• ⁢
• constant
• t
• 1
• /
• 2
• =
• Dissociation
• ⁢
• half
• -
• life
• wherein for the case of Steps E and F, the determination of the residence time between the Target and the Ligand is based on a quantitative comparison of the analytical analysis of the completely fragmented sample with the analytical analysis of the completely fragmented control sample
• and wherein for the case of Step G the determination of the residence time between the Target and the Ligand is based on a quantitative comparison of the analytical analysis of the enriched fragment sample with the analytical analysis of the enriched fragmented control sample.