Method of determining half-life of a biomolecule of interest and kit

Abstract

The present invention relates to a method of determining the half-life T1 / 2 of a biomolecule of interest, said method comprising: (a) bringing said biomolecule into contact with a compound that decreases the amount of said biomolecule; (b) determining the amount of said biomolecule as a function of time; (c) determining, from amount as a function of time, (i) the half-life T1 / 2* of said biomolecule upon said bringing into contact; and (ii) the ratio r of the equilibrium amount of said biomolecule in the presence of said compound over the equilibrium amount of said biomolecule in absence of said compound; and (d) determining said half-life as T1 / 2 = T1 / 2* / r.

Description

[0001] New EP application

[0002] Boehringer Ingelheim International GmbH

[0003] 071577.00007

[0004] METHOD OF DETERMINING HALF-LIFE OF A BIOMOLECULE OF INTEREST AND KIT

[0005] The present invention relates to a method of determining the half-life Tin of a biomolecule of interest, said method comprising: (a) bringing said biomolecule into contact with a compound that decreases the amount of said biomolecule; (b) determining the amount of said biomolecule as a function of time; (c) determining, from amount as a function of time, (i) the half-life T1 / 2* of said biomolecule upon said bringing into contact; and (ii) the ratio r of the equilibrium amount of said biomolecule in the presence of said compound over the equilibrium amount of said biomolecule in absence of said compound; and (d) determining said half-life as TI / 2 = T1 / 2’ / r.

[0006] The half-life of proteins plays a pivotal role in understanding their stability, turnover rate, and functional lifespan within a cell, which are critical factors in biological processes and assessing interventional compounds (pharmaceuticals) targeting these proteins.

[0007] Understanding protein turnover is crucial in drug discovery, particularly when assessing drug efficacy and safety. The half-life of a protein, a key aspect of protein turnover, plays a significant role in target engagement for covalent inhibitors, as well as for degraders (PROTACs, molecular glues), small molecules that induce targeted degradation of the proteins. Covalent inhibitors form a permanent bond with their target protein. Degraders do not form a permanent bond with the protein, instead, they bring an E3 ligase to a proximity of the target protein with an effect of permanent protein degradation. The duration of the target engagement (permanent bond in the case of covalent inhibitors and permanent degradation in the case of degraders) is governed by the protein's half-life. A longer half-life can lead to sustained target engagement, which can drive drug efficacy by ensuring a consistent therapeutic effect.

[0008] Prolonged target engagement also has implications for drug safety. By keeping prolonged target engagement profiles, the necessary drug concentrations for efficacy can be reduced, minimizing potential off-target effect. While this can enhance the drug's therapeutic window, it can also increase the risk of on-target toxicity due to prolonged target engagement.

[0009] In summary, quantifying protein half-life is a crucial parameter for optimizing drug efficacy and minimizing safety risks, thereby posing the need for its early assessment in the drug discovery process, best at the target identification and validation stage.

[0010] In the following, two frequently used approaches for protein half-life determination are described, namely, a "pulse" approach which introduces tracers into target proteins (e.g., SILAC method) and an approach based on complete synthesis inhibition (e.g., cycloheximide chase assay). There exist multiple implementations of these two approaches which differ in the used tracer and the synthesis inhibitor.

[0011] SILAC Method

[0012] Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a powerful method for protein half-life estimation. It involves metabolic labeling of proteins with non-radioactive, stable isotopelabeled amino acids, followed by mass spectrometry-based quantitative proteomics [1,2,3]. The SILAC method is one example of the methods of protein half-life measurements that are based on the so-called "pulse" approach, which requires the introduction of radioactive, biochemical, or stable isotope-labeled tracers into target proteins. These tracers are introduced into the target cell's metabolism either through the solvent (e.g., H₂O and hydrogen isotopes), carbon, or nitrogen metabolism (e.g.,13C-labeled carbohydrates or15N-labeled ammonia salts), or complete amino acids (e.g.,13C6-lysine or13C615N4-arginine). Tracers can then be monitored using a corresponding detection system. In the "pulse-chase" paradigm, the "pulse" is followed by a "chase" period, in which the labeled tracer is replaced, i.e., "chased" away, by an excess of the same unlabeled compound after a certain period of time. [1]

[0013] In the in-vitro SILAC method, cells are grown in media containing a 'heavy' isotopically labeled amino acid (such as13C- or15N-labeled arginine or lysine) and a 'light' non-labeled version of the same amino add. Over time, the 'heavy' amino add is incorporated into newly synthesized proteins. After an appropriate labeling period, proteins from the 'heavy' and 'light' cells are mixed and digested into peptides, which are then separated and analyzed by mass spectrometry. The 'heavy' and 'light' versions of each peptide can be distinguished based on their mass difference, and the ratio of 'heavy' to 'light' peptides provides a measure of the relative abundance of each protein in the two cell populations. Protein turnover rates are measured by monitoring the incorporation of the 'heavy' amino add over time.

[0014] Key charaderistics of the SILAC approach indude: (i) comprehensiveness: SILAC allows for the simultaneous measurement of many proteins, (ii) high accuracy: SILAC provides highly accurate and reliable quantitative data; (iii) it is versatile: SILAC can be applied to a wide range of biological systems (also in-vivo cell models); (iv) high costs: SILAC requires expensive stable isotopes and mass spectrometry equipment; (v) it is time-consuming: The process of labeling and analyzing proteins can take a significant amount of time; (vi) high degree of complexity: The data analysis for SILAC can be complex and requires specialized software; and certain limitations: Not all proteins can be labeled effectively in all cell types, particular challenge represents proteins sharing same peptides e.g. protein families (such as KRAS, NRAS, and HRAS) or wild- type and mutated proteins (such as KRAS in NCI-H727 tumor cell model). Cycloheximide Chase Assay

[0015] The cycloheximide chase assay is a common method for estimating protein half-life. It involves the use of cycloheximide, a drug that inhibits all protein synthesis in the cells. The decrease in protein levels over time is then measured, providing an estimate of protein half-life. Other synthesis inhibitors exist besides cycloheximide, for example, Actinomycin D, emetine, or puromycin, which can be used in a similar assay [4],

[0016] In a typical cycloheximide chase assay, cells are treated with cycloheximide to halt protein synthesis. At various time points following the addition of cycloheximide, samples are taken, and the protein of interest is detected and quantified, often using techniques such as Western blotting. The decrease in protein levels over time is then plotted, and the half-life of the protein is estimated from the decay curve.

[0017] Key characteristics of the cycloheximide assay include: (i) simplicity: The cycloheximide chase assay is relatively simple and straightforward to perform; (ii) versatility: It can be applied to a wide range of cell types and proteins; (iii) accessibility: The required materials, including cycloheximide, are relatively inexpensive and widely available; (iv) direct measurement: It provides a direct measurement of protein degradation rates; (v) global inhibition: Cycloheximide inhibits all protein synthesis, which can have broad effects on cell physiology and potentially confound results; (vi) toxicity: Cycloheximide is toxic to cells, limiting the duration of experiments. Using pharmacological interference (inhibitors) is inappropriate for measuring protein degradation over an extended period. The inhibitory effects cannot be separated from cytotoxicity ([4]); and (vii) inaccuracy: The method may not accurately reflect protein half-life in normal physiological conditions due to the artificial halt of protein synthesis.

[0018] In view of the shortcomings of the presently existing methods, the technical problem underlying the present invention can be seen in the provision of improved means and methods for determining the half-life of proteins and, more generally, of biomolecules.

[0019] As evidenced by the enclosed Examples, this problem has been solved by the present invention. Accordingly, in a first aspect, the present invention provides a method of determining the half-life T1 / 2 of a biomolecule of interest, said method comprising: (a) bringing said biomolecule into contact with a compound that decreases the amount of said biomolecule; (b) determining the amount of said biomolecule as a function of time; (c) determining, from amount as a function of time, (i) the halflife T1 / 2* of said biomolecule upon said bringing into contact; and (ii) the ratio r of the equilibrium amount of said biomolecule in the presence of said compound over the equilibrium amount of said biomolecule in absence of said compound; and (d) determining said half-life as TI / 2 = T1 / 2’ / r. The term "half-life" has its art-established meaning and refers to the time which elapses from synthesis of a given biomolecule of interest being completed up to the point in time where there is a 50% probability that said biomolecule has been degraded. Using ensemble terminology, it is the period of time which elapses until 50% of an ensemble of biomolecules has been degraded.

[0020] As regards the environment, the term "half-life" as used herein generally refers to a cellular environment. This may be an in vitro environment such as in cells in culture, ex vivo conditions, or an in vivo environment, for example where cells are organized in tissues, organs, and entire organisms.

[0021] Having said that, the invention is not so limited. The cellular environment is dispensable provided that the components necessary for the compound unfolding its action are present. This is straightforward in case of irreversible inhibitors but can also be implemented for PROTACs and molecular glues by providing the necessary components of the degradation machinery.

[0022] As noted above, determining half-life is an important element of the validation of a therapeutic target. Depending on where said therapeutic target is expressed in the human or animal body or to which tissue or organ a drug active on said target is to be delivered, the specific environment may differ depending on the specific case. When the half-life is to be determined using the method of the first aspect in cells such as cells in culture, said cells may be chosen to mimic the region, tissue or organ in the human or animal body where a given drug shall be delivered to and / or unfold its effect.

[0023] Said bringing into contact of step (a) is effected under conditions which allow for said compound being able to reach said biomolecule and bind to it. Such conditions prevail in cells and tissues which are indeed a preferred setting (and accordingly preferred conditions) where the method of the first aspect and the invention in general are to be practiced. As such, in one embodiment, said method is practiced in cells. In one embodiment said cells are in vitro, ex vivo, or in culture.

[0024] Having said that, also cell-free environments are envisaged. This may be buffered solutions employing established buffers such as Good's buffers. Depending on which type of compound is used (for details see further below), such cell-free environments furthermore contain those constituents which are necessary for the respective compounds to unfold their effect.

[0025] Compounds which decrease the amount of said biomolecule are not particularly limited. For example, in cellular environments, there is an equilibrium between formation and disappearance (e.g. by degradation) of said biomolecule. Any compound which converts the biomolecule into a new chemical species (e.g. by formation of a covalent bond between biomolecule and compound, the resulting adduct being a new chemical species) or removes it by other means from the equilibrium is a compound which decreases the amount of said biomolecule. Said determining amount as a function of time in step (b) can be done with any method. For example, samples may be drawn from the reaction mixture obtained in step (a) as time proceeds, and in each sample, the amount of the biomolecule of interest is determined. As regards specific methods for determining amount or concentration of biomolecules, these are subject of an embodiment disclosed further below.

[0026] It is understood that recording amount (or, equivalently, concentration) as a function of time begins at the very moment when said compound is added or before to properly define the starting concentration. Recording concentration as a function of time ends when a new equilibrium (i.e., constant) concentration becomes discernable or can be estimated from the time course recorded so far.

[0027] Once steps (a) and (b) are completed, the method of the present invention proceeds without taking recourse to a wet lab. Subsequent steps (c) and (d) may be performed manually or simply by visual inspection a plot of protein concentration vs. time or may be implemented on a computer.

[0028] Step (c) involves determining two parameters, viz. the half-life under the changed conditions (change being the addition of said compound), and the concentration of the biomolecule once a new equilibrium has established. In other words, while upon addition of the compound the concentration of the biomolecule decreases, it will gradually stop to decrease further and reach a new equilibrium, provided that concentration as a function of time is recorded for a time period of sufficient length. The length of said time period can be determined phenomenologically without further ado, i.e., by determining concentration over time until an asymptotic leveling off is seen or can be extrapolated.

[0029] The half-time T1 / 2* is the time that elapses from adding the compound until the concentration of the biomolecule has dropped to a value which is the arithmetic average of the equilibrium concentration prior to adding the compound and the new equilibrium concentration which establishes a certain time after said adding. Of note, T1 / 2* is not the inherent half-time T1 / 2 of the biomolecule in the absence of any compound as defined herein, but rather an effective or apparent half-time which governs the transition from the equilibrium prior to addition of the compound to the new equilibrium which establishes itself after addition of said compound. In other words, T1 / 2* is the decay constant governing said transition.

[0030] Given that parameter r (the other parameter to be determined) is defined as the ratio of the equilibrium concentration of said biomolecule in the presence of said compound over the equilibrium concentration of said biomolecule in absence of said compound, the effective half-time T1 / 2* is that period of time that elapses from adding the compound until the ratio of the current concentration of said biomolecule over the equilibrium concentration of said biomolecule in absence of said compound has dropped to a value of (r+l) / 2.

[0031] Determining the second parameter (r as already mentioned above) requires the concentration as a function of time displays or implies asymptotic behavior, the asymptote defining the equilibrium concentration under the new condition, i.e., in the presence of the compound.

[0032] Once both parameters in accordance with step (c) are known, the formula given in (d) provides the calculation allowing determining or estimating the half-time T1 / 2 of the biomolecule in the absence of any compound as defined herein, i.e., under undisturbed conditions.

[0033] A derivation of the formula TI / 2= T1 / 2* / r is given in the following.

[0034] It is assumed that the turnover of an endogenously expressed biomolecule assumes a zero-order synthesis and first-order degradation (see e.g. Ross et al., Molecular & Cellular Proteomics, 20 (2021), p. 100016) or Sections 2.6.1 and 3.7.2 in Gabrielsson and Weiner, Pharmacokinetic and Pharmacodynamic Data Analysis - Concepts and Applications, Apotekarsocieteten, 5th ed., 2016). If we denote a biomolecule amount (or concentration) by P, then the rate equation for the change of P over time is given by

[0035] _ dP (I)

[0036]

[0037] ^endo.synfVendo.degr> where kendo synis the rate constant of synthesis (measured in amount / concentration unit per time unit) and kendo degthe rate constant of the degradation (measured in 1 per time unit), respectively. The rate of synthesis is absolute in a sense that the same constant amount of the new biomolecule is produced per a unit of time, while the rate of degradation is fractional meaning that a kendo deg-fraction of the protein is degraded per a unit of time independently of the actual amount / concentration of the biomolecule. Both kendo synand kendo degare positive constants. Solving Equation (1) gives

[0038] p=kendo.syn+( pQ_kendo.syn\g-kendo degt (2)

[0039]

[0040] kendo.deg \ kendo degJ which is an explicit, closed formula for the time evolution of the biomolecule amount (or concentration) over time starting from the initial state PQ= P(0).

[0041] Independently of Po, the biomolecule amount / concentration will stabilize at a steady state value Pss = kendo.syn / kendo. deg exponentially fast with the rate kendo deg. Independently of whether Po> Pssor Po < Pssf the biomolecule's half-life is the time needed for P to be equal to the mid-value between Po(where P it starts) and Pss(where P eventually gets), i.e., T1 / 2satisfies P(T1 / 2) = (P0+ Pss) / 2. By introducing Pss= kendo syn / kendo deginto Equation (2), solving

[0042] + (Ro - Pss)e~k^

[0043]

[0044] degT1 / 2 =(pQ + Pss) / 2 = Pss + (Po_ pss} / 2 for T1 / 2implies that T1 / 2= ln(2)!kendo deg.

[0045] A compound-induced irreversible biomolecule modification, which is assumed to follow first-order kinetics, is modelled by introducing an additional compound concentration dependent term into Equation (1), here denoted by kcompound(c). Hence, the turnover equation (1) becomes

[0046] — - ‘fr'■endo.syn - ( \yk-endo.deg + ' ‘ k'■compound (c^ JrP ■(3)

[0047]

[0048] As stated, the compound concentration c is assumed to be constant, e.g. in in-vitro or ex-vivo experiments or in in-vivo experiments in which the compound concentration can be maintain constant for enough time.

[0049] It is assumed that before any compound administration, the protein is found stabilized in its equilibrium Pss= kendo syn / kendo deg, which is a protein amount (concentration) typically measured in the control groups. Thus, Pssis assumed to be a biomolecule baseline.

[0050] Thus, we assume that P(0) = Pssis also the initial condition for Equation (3), which has a solution similar to (2) with kendo.degreplaced by kendo.deg+ kcompound(c) wherever kendo.degappears in Equation (3). Using the same reasoning as above, we obtain that, after compound administration, the biomolecule's amount / concentration converges to a new equilibrium Ps / (c) = kendo syn / k-endo.deg + / UmpomdW) with the apparent half-life

[0051] ln(2)

[0052] 7^ / 2 (c) =

[0053]

[0054] kendo.deg T kcompound(c)

[0055] which depends on the biomolecule's concentration c.

[0056] By combining the stated terms together, we obtain

[0057] ,, k endo.de q (4)r

[0058]

[0059] (c) = =k - Tk - M T where r(c) = / ’s*s(c) / Pssis, as said, the ratio of the equilibrium concentration of said biomolecule in the presence of said compound over the equilibrium concentration of said biomolecule in the absence of said compound, in other words, r is the remaining fraction of the unmodified biomolecule at equilibrium after being exposed to said compound's concentration c.

[0060] From Equation (4), we obtain T1 / 2 = Ti / 2*(c) / r(c) for every concentration c of said compound. Without explicitly stating the dependence of the formula on the concentration, the formula is T1 / 2 = T1 / 2* / r. Figure 1 shows schematically the above-described action of said compound on said biomolecule. Of note, the actual form of kcompoundc) as a function of the compound concentration c is not important in the derivation of the formula for the compound half-life. If the time courses for multiple concentrations of a compound are measured, Equation (4) solved for kcompound(c) could further give an idea of what form kcompound(c) could have, e.g., a sigmoid curve for covalent inhibitors or a bellshaped curve for degraders exhibiting the hook effect. In the enclosed Examples, kendo deg+ kcompound( ) is denoted as / cobs(c). Thus, T*1 / 2= ln(2) / kobs(c).

[0061] In other words, the apparent half-life Tm after addition of the compound holds information about the (actual or inherent) half-life T1 / 2 prior to adding the compound, and Hr is the conversion factor.

[0062] The invention and all its aspects are characterized by distinct advantages (to be compared with the features of the prior art approaches reviewed above). In particular, and as evidenced by the Examples: (i) specificity: The method can be implemented in a highly specific manner by employing compounds targeting only one specific biomolecule such particular protein isoforms (e.g., KRAS, NRAS, HRAS) and mutant forms; (ii) versatility: It can be applied to a wide range of cell types and proteins; (iii) simplicity: The assay of the first aspect is simple and straightforward to perform, biomolecule concentration can be measured using techniques such as Western blotting. The key parameters for the protein half-life calculation can be identified even visually from the measured concentration data as a function of time; (iv) uniqueness: half-life estimated by the provided formula is independent of the compound used, and of the concentration of the used compound, i.e., no matter what compound of what concentration is used, the formula gives the same half-life, (v) direct measurement: It provides a direct measurement of degradation rates; for an increased accuracy, multiple measurements (replicates, measurements using different compounds and / or of different concentrations) can be performed.

[0063] The independency of the particular compounds used is illustrated in the Examples. For example, the half-life of KRAS G12D in GP2d cells has been determined in the presence of two different PROTACs, similarly that of MDM2 in MKN1 cells in the presence of two PROTACs, and that of KRAS G12C in NCI-H2122 by a PROTAC and two inhibitors.

[0064] While above the term "concentration" is used, the term "amount" can be used instead in the definition of the invention, its aspects and embodiments. In other words, the respective actual concentrations may be determined, but this optional. The term "amount" includes both absolute and relative amounts. Indeed, the invention can be practiced by using relative amount throughout. For example, the ratio r as defined herein is a relative amount (or relative concentration) by its very nature. Similarly, the time course of the amount of the protein of interest can be defined and determined in terms of relative amounts as well.

[0065] It is understood that accuracy and / or precision of the half-life will generally increase with the number of replicates, preferably biological replicates. In this respect it is noted that a number of Examples is based on three biological replicates (n=3; using the nomenclature of the Examples). Similarly, determining the half-life based on multiple concentrations of the compound is a further means of improving accuracy and or precision. Finally, there will be a certain dependence on the method used for determining the amounts of the protein of interest. In that respect, preference is given to proteo-mic methods such as mass spectrometry, following by (traditional) Western blotting.

[0066] If desired or deemed adequate, concentrations or amounts of biomolecules, for example of proteins, may be normalized to the total target protein amount (as measured in a control experiment), potentially further normalized to a "housekeeping" protein (such as GAPDH or Actin), or to the total cellular protein content.

[0067] In some embodiments, said biomolecule is selected from proteins, nucleic acids, and lipids; preferably proteins. These compound classes have their art-established definitions. They constitute, apart from water, a significant part of the constituents of living cells, tissues and organisms.

[0068] In some embodiments, (a) the biomolecule is a protein and said compound is selected from: (i) proteolysis-targeting chimeras (PROTACs) capable of binding to said protein; (ii) molecules capable of irreversibly binding said protein, such as an irreversible inhibitor; and (iii) molecular glues; or (b) the biomolecule is a nucleic add and said compound is a compound which decreases the amount of the nucleic acid, said compound preferably being a small inhibitory RNA, preferably selected from siRNAs, shRNAs, miRNAs, and antisense molecules; a CRISPR-associated protein with a guide RNA being bound thereto; or a nuclease; or (c) said biomolecule is a lipid and said compound is a compound which decreases the amount of said lipid, said compound preferably being a lipase.

[0069] The compounds recited in this embodiment are implementations of the above disdosed more generic notion of compounds which decrease the concentration of a biomolecule. In the embodiment above, compounds suitable for decreasing concentrations of proteins, nucleic acids and lipids, respectively, are given.

[0070] PROTACs are heterobifunctional molecules which contain a portion which binds to a protein of interest and a portion which binds to a component of the ubiquitin conjugation machinery which targets proteins for degradation, preferably an E3 ubiquitin ligase. Generally, these are small organic molecules. Upon binding to a cognate protein, this protein becomes ubiquitinated and subsequently degraded in the proteasome. A number of PROTACs have been developed, and further PROTACs may be designed with the above-described principle in mind. This applies in particular in those cases where a moiety binding to a protein of interest is known (the E3 ligase-binding moiety being essentially constant). Further information about PROTACs can be found, for example, in Luh et al., Angew Chem Int Ed Engl. 2020 Sep 1; 59(36): 15448-15466; Kofink, C. et al. 2022. " A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo." Nat. Com-mun. 13 (5969): 15pp.; and Popow, J. et al. 2023. " Targeting cancer with small molecule pan-KRAS degraders." bioRxiv. doi:10.1101 / 2023.10.24.563163. The use of PROTACs is exemplified in the Examples further below.

[0071] Irreversible inhibitors of proteins generally form a covalent bond with the cognate protein, the consequence being that the protein remains essentially permanently derivatized. In the context of proteins, irreversible inhibitors are also referred to as "suicide inhibitors".

[0072] Molecular glues are described in, for example, Li et al., doi.org / 10.3389 / fonc.2024.1401257. Molecular glues can remodel the surface of E3 ligase receptors to foster new protein-protein interactions, subsequently mediating protein degradation induced by proximity. Molecular glues directly enhance the assembly by squeezing the E3 ligase and the target protein interfaces instead of using a flexible linker like in PROTACs.

[0073] While PROTACs and molecular glues are recognized compound classes, any molecular capable of targeting a protein of interest may be used. Such molecules may be referred to as "degraders" and include but are not limited to PROTACs and molecular glues.

[0074] Irreversible inhibitors are exemplified in the Examples further below.

[0075] A number of compounds are known which degrade nucleic acid. Many of those are sequence-specific, i.e., they comprise or consist of an oligo- or polynucleotide which is similar or identical to the nucleic add to be degraded or to a part thereof. While small RNAs act on their own exploiting components of the cellular machinery (e.g. the RISC complex in case of siRNAs and shRNAs), CRISPR-associated proteins (also known as " Cas" proteins) form an assembly with a small RNA (known as guide RNA) which small RNA provides both a binding element for the Cas protein and a sequence complementary to a part of the nucleic add to be degraded.

[0076] In some embodiments, said determining of (b) is by means of Western-blotting; mass spectrometry; chromatography-based methods such as WES, and JESS; antibody-based quantification methods such as ELISA; or a luminescence-based assay such as HiBiT.

[0077] Simple Western instruments such as WES, JESS, Sally Sue or Peggy Sue are automated protein analysis platforms that combine capillary electrophoresis to separate proteins by size (CE-SDS) or charge (icIEF) with immunodetection. Simple Western assays are automatically and precisely controlled inside a capillary at the nanoliter scale. Sample is loaded into the capillary automatically, electro-phoresed and separated by size or charge as they migrate through a stacking and separation matrix. The separated proteins are then immobilized to the capillary wall via a proprietary, photoactivated capture chemistry. Target proteins are identified using a primary antibody and immunoprobed using a conjugated secondary antibody. Sample data is displayed by lane in a virtual blot like image similar to traditional Western blot results and an electropherogram view. The resulting chemiluminescent or fluorescent signals are detected and quantitated. Quantitative results such as molecular weight, signal intensity (area), percent area, and signal-to-noise for each immunodetected protein are presented in the results table automatically from Compass for Simple Western software (Bio-techne. 2024. Simple Western™ Automated Western Blot Systems, https: / / www.bio-techne.com / in-struments / simple- western).

[0078] Western blotting is a technique used to identify specific proteins from a complex mixture of proteins extracted from cells. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest. The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present; thus, doing a standard can indicate the amount of protein present (Mahmood T., Yang P. C. 2012. " Western blot: technique, theory, and trouble shooting." N Am J Med Sci. 429-434. doi:10.4103 / 1947-2714.100998).

[0079] The HiBiT assay is a protein tagging technology that offers a simple and flexible approach to detect and quantify any tagged protein. The HiBiT tag is a small, 11-amino-acid epitope tag that can produce a bioluminescent signal when bound to its complementation partner, LgBiT. The small size of HiBiT makes insertion with CRISPR efficient, and the bioluminescence method enables sensitive, quantitative detection, even of proteins with low expression (Promega. 2024. HiBiT Protein Tagging System, https: / / at.promega.com / resources / technologies / hibit-protein-tagging-system). HiBiT assays are described in, for example, Ramachandran, S., M. Szewczyk, S. H. Barghout, A. Ciulli, D. Barsyte-Lovejoy, and V. Vu. 2023. " HiBiT Cellular Thermal Shift Assay (HiBiT GETS A)." In Chemogenomics. Methods in Molecular Biology, by D. Merk and A. Chaikuad, 149-165. New York, NY: Humana. doi:10.1007 / 978-1-0716-3397-7_11.

[0080] Mass spectrometry (MS), while generally requiring more instrumentation than Western blotting, has established itself as a leading method for protein detection and quantification, including in complex samples such cellular lysates, cell culture supernatants, and cell secretion across many orders of magnitudes. WES and JESS rely on devices which provide for automated performing of Western Blots.

[0081] In some embodiments, said compound is provided at a constant or essentially constant concentration during step (b). In a number of settings, an initial administration of the compound provides for a constant or essentially constant concentration. If need be, a controlled / slow release formulation of said compound may be used, optionally in combination with an initial loading dose. In in vivo scenarios, if necessary, continuous infusion or a pump may be used.

[0082] In some embodiments, said function of time is indicative of first-order kinetics of a decrease of the amount of said biomolecule.

[0083] To explain further, if the amount (or concentration) of the biomolecule as a function of time after adding the compound follows an exponential curve, this is the time function which is observed when the kinetics of the underlying process is of first order.

[0084] Having said that, the invention is not necessarily so limited. For example, if there are indications that the kinetic is of second or higher order (for example if there are deviations from an exponential curve), it may still be attempted to just read out the time where concentration is the arithmetic average of initial and final concentration or fit a single exponential to the time course. This may lead to less accurate determinations or estimates but could still offer valuable information.

[0085] Furthermore, preferred conditions comprise zero order kinetics of the formation of the biomolecule; see also the mathematical derivation given above.

[0086] In some embodiments, (a) said function of time is fitted to an exponential function, and T1 / 2* is obtained from said exponential function; or (b) T1 / 2* is determined as the time that elapses from said bringing into contact to the point in time where the amount of said biomolecule is (r+1) / 2 times the equilibrium amount of said biomolecule in the absence of said compound.

[0087] Fitting methods are well in known the art of data modeling and data interpretation for a long time. Examples include least-squares fitting methods. As regards option (b), this can be done, in one implementation, simply by visual inspection of a plot of biomolecule amount or concentration as a function of time.

[0088] In some embodiments, said protein is extracellular, inside of or associated with a biological cell or tissue, said cell or tissue preferably being in culture.

[0089] As mentioned above, samples comprising proteins may include cell lysates, cell culture supernatants and cell secretion. In other words, a given protein of interest may be captured and determined regardless of its cellular location. In some embodiments, said method is an in vitro or ex vivo method.

[0090] The method is suitable for measuring protein half-lives in any cell system that can be cultivated in-vitro; this includes living cells and tissues taken from an organism and cultured ex- vivo. The method can also be applied for estimating protein half-lives in cells or tissues of living organisms. In that case, it is preferred that a constant concentration of the compound can be maintained over a sufficiently long period of time, and that protein concentration can be determined in the cells or tissues.

[0091] In a second aspect, the present invention provides a method of validating a biomolecule of interest as a potential therapeutic target, said method comprising applying the method of any one of the preceding claims to said protein.

[0092] As mentioned above, the duration of target engagement has implications for drug safety. By keeping prolonged target engagement profiles, the necessary drug concentrations for efficacy can be reduced, minimizing potential off-target effects. While this can enhance a drug's therapeutic window, it can also increase the risk of on-target toxicity due to prolonged target engagement. On the other hand, a very short half-life may be indicative of the respective protein of interest possibly not being the therapeutic target of first choice. Knowledge of these properties is an important element of target validation.

[0093] In addition to assessing the duration of target engagement, information on the half-life T1 / 2 of a biomolecule can be used to validate the biomolecule as a potential therapeutic target and to evaluate its pharmacological suitability. Target validation refers to demonstrating that modulation of a biomolecule, such as a protein, receptor, enzyme or RNA, is therapeutically meaningful and pharmacologically feasible. Elements considered in target validation include biological relevance, modulation feasibility, safety and selectivity, and pharmacological suitability, all of which can be assessed once the half-life of the biomolecule is known.

[0094] Biological relevance addresses the causal involvement of the biomolecule in the disease mechanism, for example through genetic association or pathway analysis. Modulation feasibility concerns whether the biomolecule can be modulated by a drug-like compound. Safety and selectivity take into account tissue expression and physiological function; in this respect, the half-life contributes to the ability of normal tissues to recover after modulation.

[0095] Pharmacological suitability directly depends on the turnover rate of the biomolecule. A short halflife may indicate rapid turnover and transient function, suggesting limited benefit from long-acting inhibition or degradation, whereas a long half-life may provide durable pharmacology but increase the risk of on-target toxicity. Incorporating half-life data into target validation enables prediction of therapeutic feasibility and early de-prioritization of targets with unfavourable turnover characteristics. The experimentally determined half-life also guides the choice of pharmacological modality. Biomolecules with long half-lives may be suited to covalent inhibitors or degraders that yield sustained activity, while short-lived biomolecules may require reversible inhibition or repeated dosing. Intermediate half-lives often represent a balanced risk-benefit profile. Furthermore, comparison of half-lives of upstream and downstream components within a signaling pathway provides insight into the expected persistence and amplitude of pharmacological responses.

[0096] A very short half-life (for example < 8h) of the biomolecule, such as that observed for proteins with rapid turnover involved in transient signaling events, may indicate that such targets are less suitable for long-acting pharmacological intervention. These proteins may require continuous exposure to reversible inhibitors or other short-acting modalities to maintain efficacy. In contrast, biomolecules with a long half-life, for example those with turnover extending over several tens of hours, may provide prolonged pharmacology and are therefore amenable to covalent inhibitors or degrader molecules that achieve durable target suppression. However, the persistence of long-lived targets also increases the likelihood of cumulative or on-target toxicity and should therefore be carefully evaluated during target selection. Knowledge of the intermediate half-life obtained by the present method thus enables prediction of the pharmacological feasibility of a biomolecule and supports early, evidence-based prioritization of therapeutic targets.

[0097] In some embodiments, said biomolecule is selected from proteins, nucleic acids, and lipids; preferably proteins.

[0098] In some embodiments, (a) the biomolecule is a protein and said compound is selected from: (i) proteolysis-targeting chimeras (PROTACs) capable of binding to said protein; (ii) molecules capable of irreversibly binding said protein, such as an irreversible inhibitor; and (iii) molecular glues; or (b) the biomolecule is a nucleic acid and said compound is a compound which decreases the amount of the nucleic acid, said compound preferably being a small inhibitory RNA, preferably selected from siRNAs, shRNAs, miRNAs, and antisense molecules; a CRISPR-associated protein with a guide RNA being bound thereto; or a nuclease; or (c) said biomolecule is a lipid and said compound is a compound which decreases the amount of said lipid, said compound preferably being a lipase. In some embodiments, said determining of (b) is by means of Western-blotting; mass spectrometry; chromatography-based methods such as WES, and JESS; antibody-based quantification methods such as ELISA; or a luminescence-based assay such as HiBiT.

[0099] In some embodiments, said compound is provided at a constant or essentially constant concentration during step (b).

[0100] In some embodiments, said function of time is indicative of first-order kinetics of a decrease of the amount of said biomolecule. In some embodiments, (a) said function of time is fitted to an exponential function, and T1 / 2* is obtained from said exponential function; or (b) T1 / 2* is determined as the time that elapses from said bringing into contact to the point in time where the amount of said biomolecule is (r+1) / 2 times the equilibrium amount of said biomolecule in the absence of said compound.

[0101] In some embodiments, said protein is extracellular, inside of or associated with a biological cell or tissue, said cell or tissue preferably being in culture.

[0102] In some embodiments, said method is an in vitro or ex vivo method.

[0103] In some embodiments, if a group of disease-relevant biomolecular targets is known or expected to exhibit little or no on-target side effects, such as toxicity, preference is given to those one or more targets within this group that display a comparatively longer inherent half-life than the remaining targets. Such preferential selection enables identification of targets that may support sustained pharmacodynamic modulation at lower compound exposure. Possible on-target side effects maybe determined, for example, by an in vitro toxicity assay using a candidate compound, such as an inhibitor and / or PROTAC, or a molecular glue as disclosed herein.

[0104] In some embodiments, if a group of disease-relevant biomolecular targets is known or expected to exhibit relevant or dose-limiting on-target side effects, preference is given to those one or more targets within this group that display a comparatively shorter inherent half-life than the remaining targets. Targets with shorter turnover may reduce the duration of pharmacodynamic engagement and thereby mitigate the risk of prolonged on-target toxicity. Possible on-target side effects may be assessed, for example, by an in vitro toxicity assay using a candidate compound, such as an inhibitor and / or a PROTAC, or a molecular glue as disclosed herein.

[0105] In a third aspect, the present invention provides a use of a compound decreasing the amount of a biomolecule for determining the half-life of said biomolecule.

[0106] In a fourth aspect, the present invention provides a use of a compound decreasing the amount of a biomolecule for determining the duration of engagement of said compound with said biomolecule. The half-life of a protein, a key parameter of protein turnover, plays an essential role in target engagement for both covalent inhibitors and small molecules that induce targeted protein degradation. The duration of target engagement is governed by the protein's half-life. Proteins with longer half-lives enable sustained target engagement, which can enhance drug efficacy by maintaining a consistent therapeutic effect. On the other hand, proteins with short half-lives rapidly replenish the pool of unmodified protein, making potential therapies more challenging. Prolonged target engagement also has safety implications: while it can reduce the required drug concentration and minimize off-target effects, it may increase the risk of on-target toxicity due to extended engagement. In drug discovery, in-vitro target-engagement assays, which measure concentration-dependent inhibition or degradation, provide valuable insight into drug potency and effect magnitude at a given assay time. Typical readouts include inhibition IC50or IC90, and degradation DC5o or DC90, together with the corresponding maximal effects (Imax or Dmax). These quantitative parameters, when combined with the measured engagement duration, enable a comprehensive assessment of compound performance by linking potency, efficacy, and kinetic persistence.

[0107] In this context, the present invention relates to the use of a compound that decreases the amount of a biomolecule for determining the duration of engagement of said compound with said biomolecule. In such a use, the compound functions as an experimental probe that provides information on how long the compound remains associated with its target and how the target level recovers once the compound is no longer present. The use comprises contacting a sample containing the biomolecule with the compound under defined conditions, then reducing or removing the compound and monitoring the amount of the biomolecule as a function of time until it approaches its pre-treatment equilibrium level. The time required for this recovery represents the duration of engagement, reflecting the persistence of the interaction between the compound and the biomolecule. Observation of this recovery behaviour enables identification of compounds displaying prolonged or transient engagement, offers an experimentally accessible measure of target-interaction kinetics, and supports selection and optimization of compounds that exhibit the desired balance between pharmacodynamic persistence, potency, and safety. In a fifth aspect, the present invention provides a kit comprising or consisting of: (a) one or more compounds selected from: (i) proteolysis-targeting chimeras (PROTACs) capable of binding to a protein of interest; (ii) irreversible inhibitors of said protein; (iii) molecular glues; (iv) compounds which decreases the amount of a nucleic acid, said compound preferably being a small inhibitory RNA, preferably selected from siRNAs, shRNAs, miRNAs, and antisense molecules; a CRISPR-associated protein and a guide RNA; or a nuclease; and (v) compounds which decrease the amount of a lipid such as a lipase; and (b) means for determining the amount of said biomolecule, preferably means for performing Western-blotting; mass spectrometry; chromatography-based methods such as WES and JESS; antibody-based quantification methods such as ELISA; or a luminescence-based assay such as HiBiT.

[0108] As such, the kit combines compositions of matter and equipment needed to perform the methods and uses of the invention.

[0109] Said kit may comprise or furthermore consist of a manual providing instructions for performing the methods and uses of the invention.

[0110] The Figures show:

[0111] Figure 1: a) Time evolution of said unmodified biomolecule amount / concentration P before and after said compound administration (indicated by a blue triangle). Before said compound is given, said biomolecule is in its equilibrium Pss(baseline). After said compound administration, said biomolecule amount P decreases from the baseline and converges to a new equilibrium Pss* with the half-time T1 / 2b) Time evolution of said unmodified biomolecule amount / concentration P normalized to the baseline, hence reaching said ratio of equilibria r.

[0112] Figure 2: Half-time of KRAS G12C in NCI-H2122 determined using covalent inhibitor AMG510 and Western blot.

[0113] Figure 3: Half-time of KRAS G12C in NCI-H2122 determined using covalent inhibitor MRTX849 and Western blot.

[0114] Figure 4: Half-time of KRAS G12C in NCI-H2122 determined using BI PROTAC A and WES.

[0115] Figure 5: Half-time of KRAS G12C in MiaPaCa-2 determined using BI PROTAC B and WES.

[0116] Figure 6: Half-time of KRAS G12D in GP2d determined using BI PROTAC B and WES.

[0117] Figure 7: Half-time of KRAS G12D in GP2d determined using BI PROTAC C and WES.

[0118] Figure 8: Half-time of KRAS G12V in Capan-2 determined using BI PROTAC A and WES.

[0119] Figure 9: Half-time of MDM2 in HEK293 determined using EX PROTAC F and EX PROTAC G, respectively, and WES.

[0120] Figure 10: Half-time of MAP4K1 in HEK293 determined using BI PROTAC D and HiBiT.

[0121] Figure 11: Half-time of MAP4K1 in HEK293 determined using BI PROTAC E and HiBiT.

[0122] Figure 12: Half-time of MDM2 in MKN1 determined using BI PROTAC F and WES.

[0123] Figure 13: Half-time of MDM2 in MKN1 determined using BI PROTAC G and WES.

[0124] The Examples illustrate the invention.

[0125] Example 1

[0126] Materials and Methods Capillary electrophoresis assay / WES

[0127] Protocol 1. 130,000-200,000 cells were seeded in 24- well plates and incubated until settled down or overnight. Compounds were dissolved in DMSO and added to cells in indicated concentrations using a digital dispenser following an optional media change. Cells were incubated for 4 or 18 hours as indicated. Medium was removed, cells were washed with PBS and lysed with 80 pl Lysis Buffer (MSD Tris Lysis Buffer (#R60TX-2) + Halt Inhibitor Cocktail (lOOx) + Benzonase 0.5 pL / ml). Samples were frozen at -80 °C and thawed at room temperature before use. Samples were transferred to a V-bottom plate and insoluble debris was pelleted by centrifugation for 5 min at maximum speed. The supernatant was transferred to a fresh plate. Master Mix and Ladder were prepared according to manufacturer's instructions for 12-230 kDa Wes Separation Module, 8 * 25 capillary cartridges, Protein Simple #SM-W004 with Anti-Rabbit Detection Module for Wes, Peggy Sue or Sally Sue, Protein Simple #DM-001. 4.8 pl lysate were mixed with 1.2 pl Master Mix to achieve a protein concentration of ~0.5 pg / pl. Protein-specific antibodies were diluted. Wes plates were prepared and run according to manufacturer's instructions. Proteins were quantified with accompanying Compass Software. Protein levels were normalised to GAPDH. Values were displayed relative to negative controls (DMSO) unless indicated otherwise.

[0128] Alternative protocol 2. Cells (50000) were seeded the day before treatment into 96-well clear-flat bottom plates and incubated overnight (37 °C, humidified tissue culture incubator, 5% CO2). Compounds were added from DMSO stock solution using a digital dispenser and cells are incubated at 37 °C as specified. At the end of the incubation, medium was removed, cells washed with ice cold PBS, PBS removed and 50 pL lysis buffer (Meso Scale Discovery #R60TX-2) supplemented with 1:100 protease inhibitor solution, 1:100 phosphatase inhibitor 1, 1:100 phosphatase inhibitor II (Meso Scale Discovery #R70AA-l), 0.5 pL / mL Benzonase (Novagen #70664-3) and 10 mM dithiothreitol added to each well. Plates with lysis buffer were immediately transferred to -80 °C freezer for at least 2 hours and then thawed on ice. Lysates were transferred to a 96 well V-bottom plate and insoluble debris was pelleted by centrifugation for 20 min. at 4000 rpm. 40 pL of the supernatant were transferred to a fresh Eppendorf PCR plate. Analysis of protein levels was conducted using the Wes System, ProteinSimple (https: / / www.bio-techne.com / ) as specified by the manufacturer. Briefly, Wes Master Mix (12-230 kDa WES Separation Module, 8 x 25capillary cartridges, Protein Simple #SM-W004) and molecular weight markers were prepared as specified in the manufacturer's recommendations. To detect primary protein-specific antibodies, anti-rabbit (Protein Simple #DM-001) or antimouse (Protein Simple #DM-002) detection modules were used. 7.2 pL lysate + 1.8 pL master mix were combined and heated to 95 °C for 5 min. Results were analyzed using the Compass Software. Capillaries with no signal were assumed to be experimental artifacts and were excluded from further analyses as indicated in the raw data. Protein levels were normalized to DMSO controls run in the same cartridge and specified in % of control.

[0129] Western blot

[0130] 5 x 105 GP5d cells in 2 mL / well were seeded into 6- well plates 24 hours before treatment. Cells were treated for 4 hours as indicated, washed with PBS and lysed with lysis buffer (1% Triton X-100, 150 mM NaCl, 1 rnM EDTA, 50 rnM Tris pH 7.4, protease inhibitor cocktail (Roche), 50 units / mL benzo-nase nuclease (Sigma). Mouse embryonic fibroblasts were seeded at 5 * 105 / mL24 hours before the treatment. They were treated with indicated compounds at 1 pM for 6 and 24 hours, then subsequently washed with PBS and lysed with the same lysis buffer as described above. Both sets of lysates were cleared by centrifugation at 4 °C, at 15800 x g for 10 min. And the supernatants stored at -20 °C. Protein concentration was determined by BCA assay (Pierce) and the absorbance at 562 nm measured by spectrophotometry on a plate reader (BMG LabtechPHERAstar). Samples were separated by SDS-PAGE using 20 pg of protein per well of NuPAGE Novex 4-12% BIS-TRIS gels (Invi-trogen) and transferred to 0.2 pm pore nitrocellulose membrane (Amersham) by wet transfer. Western blot images were obtained through detection of protein-specific antibodies using a ChemiDoc MP imaging system (Bio-Rad). Western blots were quantified using Image Studio Lite (Licor, version 5.2) with normalization to loading control and DMSO and further analyzed using GraphPad Prism (version 9.2.0) and ImageJ (Fiji).

[0131] HiBiT assay

[0132] GP5d cells expressing HiBiT-tagged protein of interest (by CRISPR-based genome engineering) were seeded at 25000 cells per well in 100 pL medium into white bottom opaque 96-well plates. Plates were incubated at 37 °C, 5% CO2 in a humidified incubator overnight. The next day, incubation medium was removed and replaced with pre-warmed fresh DMEM medium containing 10% serum from NMRI female mice (without preservatives added, sterile filtered, supplied by Envigo RMS S.r.l. Z. I.) or human serum (Sigma cat. No. S7023). Compounds (10 mM stock in DMSO) were added at logarithmic dose series using a HP Digital Dispenser D300 (Tecan), normalizing for added DMSO. Plates were further incubated at 37 °C for 18 hours. Subsequently, media was removed and replaced with 100 pL PBS at room temperature. The luminescence reaction was started by adding 100 pL of Promega Nano-Gio HiBiT lytic detection reagent mix per well, which was prepared according to the manufacturer's instructions. Plates were agitated for 15 min. on an orbital shaker and incubated for 30 min. at room temperature. Luminescence was measured on an Envision plate reader using an Ultrasensitive Luminescence Protocol for 96-well plates. Luminescence was normalized to values obtained with DMSO-treated samples and are reported as percentage of DMSO control. Lumit assay

[0133] Lumit Immunoassays use NanoBiT split-luciferase technology, where antibodies are chemically labeled with the two subunits— small bit (SmBiT) and large bit (LgBiT). When these antibodies bind to the target analyte, SmBiT and LgBiT come into proximity, forming an active luciferase and generating a luminescent signal in the presence of substrate. The small size of the NanoBiT complementation partners minimizes interference with normal protein function (2024. Lumit® Technology: Fast and Easy Immunodetection, https: / / at.promega.com / resources / technologies / lumit-biolumines-cence-immunodetection / ).

[0134] The Lumit assay quantifies endogenous (untagged / unmodified). In this assay, protein is not modified with a detection tag such as HiBiT, therefore endogenous protein synthesis and stability is unchanged.

[0135] Example 2

[0136] Results

[0137] This Example demonstrates how the relation

[0138] T1 / 2= T1 / 2* (c) / r(c)

[0139] provides experimentally grounded estimates of the inherent half-life of biomolecules across diverse systems. The datasets encompass twelve protein-cell line combinations exposed to degraders or covalent inhibitors, yielding time-dependent protein abundance profiles suitable for exponential fitting.

[0140] The inherent half-life T1 / 2, representing a compound-independent property of the biomolecule, was calculated as:

[0141] T1 / 2= T1 / 2* (c) / r(c).

[0142] Every section below is structured in the same way: first, basic information is given (biomolecule, cell line, compound name, modality, assay, and the estimated half-life), then there is reference to the Figures providing a visualization of data incl. fits with an exponential decay function, a table with estimated parameters incl. CV%, and the calculated half-life for every "qualified" fit. " Q / D" in that respect refers to "qualified" or "disqualified". The half-life of protein is calculated as an arithmetic mean of the half-lives T1 / 2 given by the formula for the qualified fits. As a qualified fit we consider the fit whose every estimated parameter has CV% below a certain CV% cut-off threshold - the lower CV%, the higher certainty in the estimated parameter. This is aimed to serve as a criterion of fit quality. The CV%-cut-off threshold may be different for different examples - in principle, the higher data density, the lower cut-off threshold is used (typically 20% or 30% CV). If a fit does not qualify for the half-life calculation, this is indicated in the table by D and the corresponding T1 / 2 is labelled as NA. Otherwise, the qualified fit is labelled by Q and Tishas a numeric value calculated from the formula.

[0143] If more compounds are used to determine the half-life T1 / 2 of the same protein in the same cell line, the "final" half-life can be calculated as a mean of the half-lives from all qualified fits for all compounds.

[0144] (1) KRAS G12C in NCI-H2122 cells treated with AMG510

[0145] Short summary

[0146] • Protein: KRAS G12C

[0147] • Cell line: NCI-H2122

[0148] • Compound: AMG510 (clinical compound by Amgen)

[0149] • Modality: Covalent inhibitor

[0150] • Assay: Western blot

[0151] • Estimated half-life: 13.9 ± 1.54 hours

[0152] • CV% cut-off: 20%

[0153] • Number of replicates per used concentration: 3

[0154] The structure of AMG510 is shown below.

[0155]

[0156] Qualified fits were obtained at 10 nM, 100 nM, and 1000 nM as shown in Table 1. All three concentrations produced consistent estimates of TI / 2*(C) and r(c), yielding a uniform inherent half-life for KRAS G12C.

[0157] Decay curves and fits are shown in Figure 2.

[0158] Table 1: Parameter estimates for KRAS G12C in NCI-H2122 cells (AMG510)

[0159] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D Tl / 2

[0160] 10.00 0.0972 (5.8) 1 (fixed) 0.59 (1.2) Q 12.18

[0161] 100.00 0.383 (10) 1 (fixed) 0.13 (8.4) Q 14.35

[0162] 1000.00 1.05 (12.4) 1 (fixed) 0.04 (13.9) Q 15.16

[0163] Calculated half-life from qualified fits: 13.9 ± 1.54 hours.

[0164] (2) KRAS G12C in NCI-H2122 cells treated with MRTX849

[0165] Short summary

[0166] • Protein: KRAS G12C

[0167] • Cell line: NCI-H2122

[0168] • Compound: MRTX849 (clinical compound by Mirati)

[0169] • Modality: Covalent inhibitor

[0170] • Assay: Western blot

[0171] • Estimated half-life: 9.55 ± 5.25 hours

[0172] • CV% cut-off: 20%

[0173] • Number of replicates per used concentration: 3

[0174] The structure of MRTX849 is shown below:

[0175]

[0176] MRTX849 Qualified fits were obtained at 10, 100, and 1000 nM, with model parameters shown in Table 2. As with AMG510, the inherent half-life derived from these datasets was consistent across concentrations and similar to the value obtained in section (1).

[0177] Decay curves and fits are shown in Figure 3.

[0178] Table 2: Parameter estimates for KRAS G12C in NCI-H2122 cells (MRTX849)

[0179] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D Tl / 2

[0180] 10.00 0.187 (15.9) 1 (fixed) 0.79 (1.3) Q 4.70

[0181] 100.00 0.183 (8.7) 1 (fixed) 0.25 (4.8) Q 15.12

[0182] 1000.00 0.692 (8.4) 1 (fixed) 0.11 (6.5) Q 8.83

[0183] Calculated half-life from qualified fits: 9.55 ± 5.25 hours.

[0184] (3) KRAS G12C in NCI-H2122 cells treated withBI PROTAC A

[0185] Short summary

[0186] • Protein: KRAS G12C

[0187] • Cell line: NCI-H2122

[0188] • Compound: BI PROTAC A (undisclosed)

[0189] • Modality: PROTAC

[0190] • Assay: WES

[0191] • Estimated half-life: 13.33 ± 1.05 hours

[0192] • CV% cut-off: 30%

[0193] • Number of replicates per used concentration: 1

[0194] Qualified fits were obtained at 62 nM and 250 nM, while the fit at 1000 nM did not meet the CV % criteria and was therefore disqualified, as summarised in Table 3. The resulting inherent half-lives matched those derived from the covalent inhibitors, confirming modality independence.

[0195] Decay curves and fits are shown in Figure 4. Table 3: Parameter estimates for KRAS G12C in NCI-H2122 cells (BI PROTAC A)

[0196] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2 62 0.148 (11) 1.56 (5.5) 0.33 (4.4) Q 14.07 250 0.247 (27.3) 1.31 (20.3) 0.22 (12.2) Q 12.58 1000 0.0812 (53) 1 (32.7) 0.06 (91.2) D NA Calculated half-life from qualified fits: 13.33 ± 1.05 hours.

[0197] The half-life of KRAS G12C calculated from all qualified fits for all three compounds (AMG510, MRTX849 and PROTAC A) is 12.12 ± 3.4 hours.

[0198] (4) KRAS G12C in MiaPaCa-2 cells with BI PROTAC B

[0199] Short summary

[0200] • Protein: KRAS G12C

[0201] • Cell line: MiaPaCa-2

[0202] • Compound: BI PROTAC B (undisclosed)

[0203] • Modality: PROTAC

[0204] • Assay: WES

[0205] • Estimated half-life: 9.4 ± 2.3 hours

[0206] • CV% cut-off: 20%

[0207] • Number of replicates per used concentration: 3

[0208] Qualified fits were obtained at 3.70, 11.10, and 33.30 nM as shown in Table 4. Fits at lower (1.23 nM) and higher (100 nM, 300 nM) concentrations did not meet the CV % threshold. The resulting T1 / 2 was comparable to that observed in NCI-H2122 cells.

[0209] Decay curves and fits are shown in Figure 5.

[0210] Table 4. Parameter estimates for KRAS G12C in MiaPaCa-2 cells (BI PROTAC B)

[0211] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0212] 1.23 0.166 (39.6) 1.07 (3.1) 0.7 (7) D

[0213]

[0214] 3.70 0.232 (14) 1.16 (3.3) 0.42 (5.8) Q 7.09 Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0215] 11.10 0.272 (10) 1.22 (3.8) 0.27 (6.3) Q 9.42

[0216] 33.30 0.271 (13.3) 1.16 (5.7) 0.22 (9.4) Q 11.70

[0217] 100.00 0.171 (16.2) 1.15 (5.3) 0.15 (21.5) D NA

[0218] 300.00 0.0757 (32.8) 1.18 (3.8) 0 (Inf) D NA

[0219] Calculated half-life from qualified fits: 9.4 ± 2.3 hours.

[0220]

[0221] (5) KRAS G12D in GP2d cells treated with BI PROTAC B

[0222] Short summary

[0223] • Protein: KRAS G12D

[0224] • Cell line: GP2d

[0225] • Compound: BI PROTAC B (undisclosed)

[0226] • Modality: PROTAC

[0227] • Assay: WES

[0228] • Estimated half-life: 22.03 ± 1.95 hours

[0229] • CV% cut-off: 20%

[0230] • Number of replicates per used concentration: 3

[0231] Qualified fits were obtained at 11.10 nM, 33.30 nM, and 100.00 nM, as shown in Table 5. Lower concentrations (1.23 nM and 3.70 nM) and the highest 300.00 nM did not meet the CV% quality criteria and were therefore excluded from half-life calculation. All qualified fits yielded similar inherent half-lives.

[0232] Decay curves and fits are shown in Figure 6.

[0233] Table 5. Parameter estimates for KRAS G12D in GP2d cells (BI PROTAC B)

[0234] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0235] 1.23 0.179 (55.5) 1.08 (6.6) 0.63 (11.4) D

[0236]

[0237] 3.70 0.432 (23) 1.33 (9.2) 0.43 (7.2) D NA

[0238] 11.10 0.219 (16.1) 1.09 (7.6) 0.15 (15.4) Q 20.81 Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0239] 33.30 0.324 (8.5) 1.24 (6.4) 0.1 (9.1) Q 21.00

[0240] 100.00 0.293 (9.9) 1.18 (6.4) 0.1 (9.8) Q 24.27

[0241] 300.00 0.088 (57.3) 0.9 (9.8) 0 (Inf) D NA

[0242] Calculated half-life from qualified fits: 22.03 ± 1.95 hours.

[0243]

[0244] (6) KRAS G12D in GP2d cells treated with BI PROTAC C

[0245] Short summary

[0246] • Protein: KRAS G12D

[0247] • Cell line: GP2d

[0248] • Compound: BI PROTAC C

[0249] • Modality: PROTAC

[0250] • Assay: WES

[0251] • Estimated half-life: 18.03 ± 7.21 hours

[0252] • CV% cut-off: 20%

[0253] • Number of replicates per used concentration: 3

[0254] The structure of BI PROTAC C is shown below:

[0255]

[0256] BI PROTAC C produced a clear degradation response against KRAS G12D in GP2d cells. Qualified fits were obtained at 11.10, 33.30, 100.00, and 300.00 nM, as summarised in Table 6. Fits at 1.23 and - 7-

[0257] 3.70 nM were disqualified. The inherent half-life was consistent with the value obtained from PROTAC B.

[0258] Decay curves and fits are shown in Figure 7.

[0259] Table 6. Parameter estimates for KRAS G12D in GP2d cells (BI PROTAC C)

[0260] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0261] 1.23 0.431 (48.8) 1.35 (9.5) 0.82 (6) D NA

[0262] 3.70 0.247 (22.5) 1.39 (5.9) 0.54 (7.6) D NA

[0263] 11.10 0.377 (10.9) 1.37 (6) 0.25 (6.2) Q 7.34

[0264] 33.30 0.359 (11.3) 1.09 (8.7) 0.09 (11.3) Q 20.79

[0265] 100.00 0.562 (9.3) 1.18 (9.9) 0.05 (9.8) Q 23.15

[0266] 300.00 0.837 (12) 1.24 (16.1) 0.04 (12) Q 20.82

[0267] Calculated half-life from qualified fits: 18.03 ± 7.21 hours

[0268]

[0269] (7) KRAS G12V in Capan-2 cells treated with BI PROTAC A

[0270] Short summary

[0271] • Protein: KRAS G12V

[0272] • Cell line: Capan-2

[0273] • Compound: BI PROTAC A (undisclosed)

[0274] • Modality: PROTAC

[0275] • Assay: WES

[0276] • Estimated half-life: 11.07 ± 1.32 hours

[0277] • CV% cut-off: 30%

[0278] • Number of replicates per used concentration: 4

[0279] Qualified fits were obtained at 15.60, 62.50, and 250.00 nM, as shown in Table 7. The fit at 1000 nM was disqualified. The inherent half-life derived from qualified fits was consistent across concentrations. Decay curves and fits are shown in Figure 8.

[0280] Table 7. Parameter estimates for KRAS G12V in Capan-2 cells (BI PROTAC A)

[0281] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0282] 15.60 0.229 (22.6) 1.24 (12.6) 0.27 (8.8) Q 11.24 62.50 0.302 (16) 1.25 (11.5) 0.19 (7.3) Q 12.29 250.00 0.294 (25.2) 1.09 (15.2) 0.24 (9.2) Q 9.67 1000.00 0.168 (50) 1 (15) 0.39 (11) D

[0283]

[0284] Calculated half-life from qualified fits: 11.07 ± 1.32 hours.

[0285] (8) MDM2 in HEK293 cells treated with EX PROTACS F and G

[0286] Short summary

[0287] • Protein: MDM2

[0288] • Cell line: HEK293

[0289] • Compounds: EX PROTACs F and G (compounds by Kymera)

[0290] • Modality: PROTAC

[0291] • Assay: WES

[0292] • Estimated half-life: 1.15 ± 0.48 hours

[0293] • CV% cut-off: not applied

[0294] • Number of replicates per used concentration: 2

[0295] The structure of EX PROTAC F is shown below:

[0296]

[0297] Each compound was evaluated at a single concentration (100 nM), as shown in Table 8. As only one time course per compound was available, no qualification filtering was applied. Both datasets yielded comparable T1 / 2 *(c) and r(c), producing consistent inherent half-life estimates for MDM2, a protein known for rapid turnover.

[0298] Decay curves and fits are shown in Figure 9.

[0299] Table 8. Parameter estimates for MDM2 in HEK293 cells (EX PROTACs F and G)

[0300] Drug Cone (nM) kobs (CV%) top (CV%) bottom (CV%) T1 / 2 PROTAC F 100.00 2.78 (26.5) 1 (fixed) 0.31 (9.5) 0.81 PROTAC G 100.00 2.25 (12.3) 1 (fixed) 0.21 (7.3) 1.49 Calculated half-life from the fits: 1.15 ± 0.48 hours.

[0301]

[0302] Additional remarks: In this example, we have tested one concentration (100 nM) of two external PROTACs (by Kymera) internally labelled as PROTAC F and PROTAC G. In this case we do not apply any quality fit criteria since we have only one time course per compound. The estimated halflife is a mean of the calculated half-lives for both compounds.

[0303] It is known in the literature that MDM2 is an unstable protein. For example, the map of short-lived proteins available at harvard.edu reports 0.65h half-life of MDM2 in HEK293T cells as determined by a CHX experiment, while 2.5h half-life of MDM2 in HEK293T cells is reported in [5],

[0304] (9) MAP4K1 in HEK293 cells treated with BI PROTAC D

[0305] Short summary

[0306] • Protein: MAP4k1

[0307] • Cell line: HEK293

[0308] • Compound: BI PROTAC D (undisclosed)

[0309] • Modality: PROTAC

[0310] • Assay: HiBiT

[0311] • Estimated half-life: 2.51 ± 1.22 hours

[0312] • CV% cut-off: 20%

[0313] • Number of replicates per used concentration: 2 Qualified fits were obtained across the tested concentration range from 1.37 to 333 nM, summarized in Table 9. The resulting inherent half-lives were uniform across the entire concentration range.

[0314] Decay curves and fits are shown in Figure 10.

[0315] Table 9. Parameter estimates for MAP4K1 in HEK293 cells (BI PROTAC D)

[0316] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0317] 1.37 0.223 (18.4) 1 (fixed) 0.71 (2.3) Q 4.36

[0318] 4.12 0.391 (10.4) 1 (fixed) 0.5 (2.2) Q 3.57

[0319] 12.30 0.951 (8.4) 1 (fixed) 0.35 (2.1) Q 2.06

[0320] 37.00 2.08 (7.1) 1 (fixed) 0.25 (2.1) Q 1.33

[0321] 111.00 2.56 (7.1) 1 (fixed) 0.2 (2.4) Q 1.36

[0322] 333.00 1.36 (5.5) 1 (fixed) 0.21 (2.1) Q 2.39

[0323] Calculated half-life from qualified fits: 2.51 ± 1.22 hours.

[0324]

[0325] (10) MAP4K1 in HEK293 cells treated with BI PROTAC E

[0326] Short summary

[0327] • Protein: MAP4k1

[0328] • Cell line: HEK293

[0329] • Compound: BI PROTAC E (undisclosed)

[0330] • Modality: PROTAC

[0331] • Assay: HiBiT

[0332] • Estimated half-life: 2.41 ± 0.66 hours

[0333] • CV% cut-off: 20%

[0334] • Number of replicates per used concentration: 2 Qualified fits were obtained across the tested concentration range from 1.37 to 333 nM, and fitted parameters are summarized in Table 10. The inherent half-life was consistent with that obtained from PROTAC D, confirming reproducibility across degraders.

[0335] Decay curves and fits are shown in Figure 11.

[0336] Table 10. Parameter estimates for MAP4K1 in HEK293 cells (BI PROTAC E)

[0337] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0338] 1.37 0.489 (18.3) 1 (fixed) 0.73 (1.5) Q 1.93

[0339] 4.12 0.82 (8.8) 1 (fixed) 0.52 (1.4) Q 1.63

[0340] 12.30 0.904 (5.9) 1 (fixed) 0.37 (1.4) Q 2.05

[0341] 37.00 0.904 (5.4) 1 (fixed) 0.31 (1.6) Q 2.49

[0342] 111.00 0.725 (4.1) 1 (fixed) 0.31 (1.3) Q 3.10

[0343] 333.00 0.58 (6.6) 1 (fixed) 0.37 (1.8) Q 3.28

[0344] Calculated half-life from qualified fits: 2.41 ± 0.66 hours.

[0345]

[0346] (11) MDM2 in MKN1 cells treated with EX PROTAC F

[0347] Short summary

[0348] • Protein: MDM2

[0349] • Cell line: MKN1

[0350] • Compounds: EX PROTAC F (compound by Kymera)

[0351] • Modality: PROTAC

[0352] • Assay: WES

[0353] • Estimated half-life: 1.1 ± 0.36 hours

[0354] • CV% cut-off: 30%

[0355] • Number of replicates per used concentration: 3 For MDM2 in MKN1 treated with EX PROTAC F, qualified fits were obtained at 16 nM and 62 nM, while the fit at 250 nM, was disqualified as shown in Table 11. Both qualified fits produced similar inherent half-life estimates.

[0356] Decay curves and fits are shown in Figure 12.

[0357] Table 11. Parameter estimates for MDM2 in MKN1 cells (EX PROTAC F)

[0358] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D Tl / 2

[0359] 16.00 1.77 (28) 1 (fixed) 0.29 (14.5) Q 1.35

[0360] 62.00 3.34 (22.6) 1 (fixed) 0.24 (11.2) Q 0.85

[0361] 250.00 7.98 (40.3) 1 (fixed) 0.18 (16.5) D NA

[0362] Calculated half-life from the fits: 1.1 ± 0.36 hours.

[0363]

[0364] (12) MDM2 in MKN1 cells treated with EX PROTAC G

[0365] Short summary

[0366] • Protein: MDM2

[0367] • Cell line: MKN1

[0368] • Compounds: EX PROTAC G (compound by Kymera)

[0369] • Modality: PROTAC

[0370] • Assay: WES

[0371] • Estimated half-life: 0.81 ± 0.53 hours

[0372] • CV% cut-off: 30%

[0373] • Number of replicates per used concentration: 3

[0374] For MDM2 in MKN1 treated with EX PROTAC G, qualified fits were obtained at 62 nM and 250 nM, while the fit at 16 nM was disqualified, as summarised in Table 12. The resulting inherent half-lives were consistent with those from EX PROTAC F.

[0375] Decay curves and fits are shown in Figure 13. Table 12. Parameter estimates for MDM2 in MKN1 cells (EX PROTAC G)

[0376] Cone (nM) kobs (CV%) top (CV%) bottom (CV%) Q / D T1 / 2

[0377] 16.00 0.934 (30.4) 1 (0) 0.45 (12) D

[0378]

[0379] 62.00 2.06 (24) 1 (0) 0.28 (12.2) Q 1.19

[0380] 250.00 5.06 (28.5) 1 (0) 0.31 (9.5) Q 0.44

[0381] Calculated half-life from the fits: 0.81 ± 0.53 hours.

[0382]

[0383] Summary

[0384] Across all twelve systems analysed, the parameters TI / 2*(C) and r(c) were consistently obtainable from the qualified exponential fits. Despite differences in biochemical mechanism (covalent inhibition vs. degradation), assay modality (Western blot, WES, HiBiT), and concentration range, the resulting inherent half-life T1 / 2 remained stable for each biomolecule within a given cell line.

[0385] This demonstrates that T1 / 2 is an intrinsic property of the biomolecule, independent of the perturbing compound and independent of concentration.

[0386] The robustness observed across modalities and datasets provides the experimental foundation for the cross-dataset consistency demonstrated in Example 3.

[0387] Example 3

[0388] Cross-dataset validation of the inherent half-life calculation

[0389] This Example assesses the reproducibility of the inherent half-life T1 / 2 across the twelve datasets described in Example 2. For each dataset, TI / 2*(C) and r(c) were extracted from qualified fits and used to calculate Tm. Inter-modality consistency

[0390] KRAS G12C in NCI-H2122 yielded comparable inherent half-lives for two covalent inhibitors (AMG510 and MRTX849) and one degrader (BI PROTAC A), despite pronounced differences in maximal effect and decay kinetics. This confirms that T1 / 2 is independent of compound modality. Inter-compound consistency

[0391] KRAS G12D in GP2d produced nearly identical inherent half-lives from BI PROTACs B and C, even though their degradation efficiencies differed. A similar pattern was observed for MAP4K1 and MDM2 datasets with distinct PROTACs.

[0392] Cell-line dependence

[0393] MDM2, a short-lived protein, showed consistently short inherent half-lives in both HEK293 and MKN1 cells, with expected differences between the two cell lines reflecting biological background rather than compound effects.

[0394] Conclusion

[0395] Across all systems, the fitted parameters TI / 2*(C) and r(c) reliably yielded a consistent inherent halflife T1 / 2, confirming that T1 / 2 is an intrinsic and modality-independent property of the biomolecule. The broad reproducibility across compounds, modalities, cell lines, and assay formats demonstrates the robustness and practical utility of the method for target evaluation, pharmacological profiling, and early drug discovery. Further References

[0396] [1] A. B. Ross, J. D. Langer, & M. Jovanovic, Proteome Turnover in the Spotlight: Approaches, Applications, and Perspectives. Mol. Cell. Proteom. 20 (2021), 100016.

[0397] [2] S. E. Ong, B. Blagoev, I. Kratchmarova, D. B. Kristensen, H. Steen, A. Pandey, & M. Mann, Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Molecular & Cellular Proteomics 1(5) (2002), pp. 376-386.

[0398] [3] M. K. Doherty, D. E. Hammond, M. J. Clague, S. J. Gaskell, & R. J. Beynon, Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC, Journal of Proteome Research 8(1) (2009), pp. 104-112.

[0399] [4] C. Chan, P. Martin, N. J. Liptrott, M. Siccardi, L. Almond, & A. Owen, Incompatibility of chemical protein synthesis inhibitors with accurate measurement of extended protein degradation rates, Pharma Res Per 5(5) (2017), e00359.

[0400] [5] R. Genolet, G. Rahim, P. Gubler-Jaquier, & J. Curran, The translational response of the human mdm2 gene in HEK293T cells exposed to rapamycin: a role for the 5'-utrs, Nucleic Acids Research 39 (2010), pp. 989-1003.

Claims

Claims1. A method of validating a biomolecule of interest as a potential therapeutic target, comprising applying a method of determining the half-life T1 / 2 of a biomolecule of interest to said protein, said method comprising(a) bringing said biomolecule into contact with a compound that decreases the amount of said biomolecule;(b) determining the amount of said biomolecule as a function of time;(c) determining, from amount as a function of time,(i) the half-life T1 / 2* of said biomolecule upon said bringing into contact; and(ii) the ratio r of the equilibrium amount of said biomolecule in the presence of said compound over the equilibrium amount of said biomolecule in absence of said compound; and(d) determining said half-life as T1 / 2 = T1 / 2* / r.

2. A method of determining the half-life T1 / 2 of a biomolecule of interest, said method comprising:(a) bringing said biomolecule into contact with a compound that decreases the amount of said biomolecule;(b) determining the amount of said biomolecule as a function of time;(c) determining, from amount as a function of time,(j) the half-life T1 / 2* of said biomolecule upon said bringing into contact; and(iii) the ratio r of the equilibrium amount of said biomolecule in the presence of said compound over the equilibrium amount of said biomolecule in absence of said compound; and(d) determining said half-life as T1 / 2 = T1 / 2* / r.

3. The method of claim 1 or 2, wherein said biomolecule is selected from proteins, nucleic acids, and lipids; preferably proteins.

4. The method any one of claims 1 to 3, wherein(a) the biomolecule is a protein and said compound is selected from:(i) proteolysis-targeting chimeras (PROTACs) capable of binding to said protein; (ii) molecules capable of irreversibly binding said protein, such as an irreversible inhibitor; and(iii) molecular glues; or(b) the biomolecule is a nucleic acid and said compound is a compound which decreases the amount of the nucleic acid, said compound preferably being a small inhibitory RNA, preferably selected from siRNAs, shRNAs, miRNAs, and antisense molecules; a CRISPR- associated protein with a guide RNA being bound thereto; or a nuclease; or(c) said biomolecule is a lipid and said compound is a compound which decreases the amount of said lipid, said compound preferably being a lipase.

5. The method of any one of claims 1 to 4, wherein said determining of (b) is by means of Western-blotting; mass spectrometry; chromatography-based methods such as WES, and JESS; antibody-based quantification methods such as ELISA; or a luminescence-based assay such as HiBiT.

6. The method of any one of claims 1 to 5, wherein said compound is provided at a constant or essentially constant concentration during step (b).

7. The method of any one of the claims 1 to 6, wherein said function of time is indicative of first- order kinetics of a decrease of the amount of said biomolecule.

8. The method of any one of claims 1 to 7, wherein(a) said function of time is fitted to an exponential function, and T1 / 2* is obtained from said exponential function; or(b) T1 / 2* is determined as the time that elapses from said bringing into contact to the point in time where the amount of said biomolecule is (r+1) / 2 times the equilibrium amount of said biomolecule in the absence of said compound.

9. The method of any one of claims 1 to 8, wherein said protein is extracellular, inside of or associated with a biological cell or tissue, said cell or tissue preferably being in culture.

10. The method of any one of claims 1 to 9, wherein said method is an in vitro or ex vivo method.

11. Use of a compound decreasing the amount of a biomolecule for determining the half-life of said biomolecule.

12. Use of a compound decreasing the amount of a biomolecule for determining the duration of engagement of said compound with said biomolecule.

13. A kit comprising or consisting of:(a) one or more compounds selected from:(i) proteolysis-targeting chimeras (PROTACs) capable of binding to a protein of interest;(ii) irreversible inhibitors of said protein;(iii) molecular glues;(iv) compounds which decreases the amount of a nucleic acid, said compound preferably being a small inhibitory RNA, preferably selected from siRNAs, shRNAs, miRNAs, and antisense molecules; a CRISPR-associated protein and a guide RNA; or a nuclease; and(v) compounds which decrease the amount of a lipid such as a lipase; and(a) means for determining the amount of said biomolecule, preferably means for performing Western-blotting; mass spectrometry; chromatography-based methods such as WES and JESS; antibody-based quantification methods such as ELISA; or a luminescence-based assay such as HiBiT.

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