discovery poster
ChameLogK: A Rapid, High-Throughput Assay to AssessChameLogK: A Rapid, High Throughput Assay to Assess Bioavailability of Beyond Rule of Five CompoundsBioavailability of Beyond Rule of Five Compounds
1,2 e StaceCatherine StaceDharmesh BhattDharmesh Bhatt, Andy Morley , Claudio Dagostin, Andy Morley , Claudio Dagostin, Mayank Gupta , Pranav Pandya, Mayank Gupta , Hardic Dave, Pranav Pandya, Dhruv Lad, Hardic Dave, Vinkal Zalavadia, Dhruv Lad , Vinkal Zalavadia, Vishal Patel , Hemal Soni, Vishal Patel, Nilesh Dagia, Hemal Soni, Sunil Shah, Nilesh Dagia, Prashant Shah, Sunil Shah , Prashant Shah
overy Pvt. Ltd., Changodar, Ahmedabad, India.o2h Discovery Pvt. Ltd., Changodar, Ahmedabad, India.o2h Ltd., Mill SciTech Park, Hauxton, Cambridgeshire, UK.o2h Ltd., Mill SciTech Park, Hauxton, Cambridgeshire, UK.* Correspondence to [email protected]* Correspondence to [email protected] act Abstract
LogKChameLogK is a cutting-edge method for measuring chameleonicity, a vital factor in assessing cell permeability propensity of small molecules, includingis a cutting edge method for measuring chameleonicity, a vital factor in assessing cell permeability propensity of small molecules, including d Rule‘Beyondof Five’Rule-of-such asFive’PROTACs,such as PROTACs, macrocycles and peptides. Chameleonicity is ability of molecules to conformationally adapt to their environment,macrocycles and peptides Chameleonicity is ability of molecules to conformationally adapt to their environment, IAM
IAM by intramoleculardriven by intramolecular hydrogen bonds; ChameLogK combines lipophilicity (BRlogD) and polarityhydrogen bonds ChameLogK combines lipophilicity (BRlogD) and polarity (Δlog kw
) descriptors. Leveraging literature methods,Leveraging literature methods, s developedo2h has developed a reliable, high-throughput platform to rapidly and inexpensively measure chameleonicity, accelerating optimization of Beyond Rule-of-a reliable, high throughput platform to rapidly and inexpensively measure chameleonicity, accelerating optimization of Beyond Rule of erapeuticFive therapeutic compoundscompounds meleonicity: An indicator of bioavailability and cellChameleonicity: An indicator of bioavailability and cell
Assay Validation and Platform DevelopmentAssay Validation and Platform Development permeability for Beyond Rule of Five moleculespermeability for Beyond Rule of Five molecules o2h conducted a rigorous validation process, with a variety of different chemicalo2h conducted a rigorous validation process, with a variety of different chemical eleonicity is the capacity of aChameleonicity is the capacity of a ule to hide polarity in nonmolecule to hide polarity in non-polarpolar nments and expose it in water,environments and expose it in water, chieving sufficient permeabilityhelp achieving sufficient permeability olubility for drug molecules withand solubility for drug molecules with olecular weight [1]high molecular weight [1]
species, underscoring the reliability of the assay as a robust and transferable platform,species, underscoring the reliability of the assay as a robust and transferable platform, broadly applicable for multiple drug discovery programs.broadly applicable for multiple drug discovery programs.
Validated with >20 different compounds, including macrocycles, nonValidated with >20 different compounds, including macrocycles, non-macrocyclicmacrocyclic compounds, and PROTACscompounds, and PROTACs >100 compounds examined to date confirm versatility and effectiveness in designing>100 compounds examined to date confirm versatility and effectiveness in designing bioavailable compounds beyond Ro5 constraintsbioavailable compounds beyond Ro5 constraints
Adapted from DanielusAdapted from Danieluset al, Nature 2020 [2] et al, Nature 2020 [2]
A any years, it has been known that bRo5 molecules, such as macrocycles and smallFor many years, it has been known that bRo5 molecules, such as macrocycles and small
B es, can exhibit chameleonic propertiespeptides, can exhibit chameleonic propertiesconformational flexibilityconformational flexibility - driven by thedriven by the tion of intramolecular hydrogen bondsformation of intramolecular hydrogen bonds [3][3]
ompounds often suffer from a poor permeability and bioavailability profile.bRo5 compounds often suffer from a poor permeability and bioavailability profile. sign and optimization of compounds with chameleonicThe design and optimization of compounds with chameleonic rties is central toproperties is central to optimizing solubility and permeability [4]optimizing solubility and permeability [4]
IAM
IAM
D, Δlog kBRlogD, Δw and ChameLogK are threelog kw and ChameLogK are three ementary measures revealing lipophilicity,complementary measures revealing lipophilicity, lity, exposed polarity, and chameleonic masking;solubility, exposed polarity, and chameleonic masking; er forming a triad of drugtogether forming a triad of drug-likeness, guidinglikeness, guiding er compound prioritization by explainingsmarter compound prioritization by explaining ability and bioavailability [5]permeability and bioavailability [5]
Figure 1: A. Output data showing correlation of Log K’ with % acetonitrile for a PROTAC andFigure 1: A. Output data showing correlation of Log K’ with % acetonitrile for a PROTAC and a classic Ro5 compound;a classic Ro5 compound;B. Summary of chameleonicity across a range of chemical speciesB. Summary of chameleonicity across a range of chemical species o2h has streamlinedo2h has streamlined
Results processes for processes for PROTACs: Displayed distinct “Chameleonic” qualities.PROTACs: Displayed distinct “Chameleonic” qualities.maximal throughputmaximal throughput establishedOther established phys chem measures relating to permeability, such as ePSA [6][1] arephys chem measures relating to permeability, such as ePSA [6][1] are aratively low throughput and costly, revealing a need for an easilycomparatively low throughput and costly, revealing a need for an easily-accessibleaccessible and productivityand productivity
Non macrocyclesNon-macrocycles: Moderate chameleonicity, showingModerate chameleonicity, showing ative measure to support rapid design and optimization of bRo5 compoundsalternative measure to support rapid design and optimization of bRo5 compounds expected flexibility compared to classic Ro5 and PROTACsexpected flexibility compared to classic Ro5 and PROTACscreating a cost-creating a cost- Macrocycles: Led by Cyclosporin, showed variable behaviourMacrocycles: Led by Cyclosporin, showed variable behavioureffective and easy-effective and easy- [5] and hasE3 Ligands: Consistent behaviour, possibly involving internalE3 Ligands: Consistent behaviour, possibly involving internalaccess version of thisaccess version of this ameLogK method was pioneered in byThe ChameLogK method was pioneered in by GiuliaGiulia Caron’s group in TurinCaron’s group in Turin[5] and has ground breakingground-breaking folding or interactionsfolding or interactions red significant interest in the academic settinggarnered significant interest in the academic setting, but had not been developed into, but had not been developed into technique
Ro5 moleculesRo5 molecules like caffeine, and diazepam defied expectationlike caffeine, and diazepam defied expectation technique transferable platform for routine screening in the commercial settinga fully-transferable platform for routine screening in the commercial setting scovery is the first in the world to offer the ChameLogK assay to clients too2h discovery is the first in the world to offer the ChameLogK assay to clients to rt their drug discovery programssupport their drug discovery programs
Supporting a range of modalitiesSupporting a range of modalities
Measuring ChameLogK: How it worksMeasuring ChameLogK: How it works
The increasing need to explore the bRo5 spaceThe increasing need to explore the bRo5 spaceto access novel chemistry, betterto access novel chemistry, better mechanisms of action, and exciting new modalities, requires a way to quickly andmechanisms of action, and exciting new modalities, requires a way to quickly and inexpensively measure chameleonicityinexpensively measure chameleonicity
LogK is the difference (ChameLogK is the difference (Δlog k′) between theΔlog k′) between the ental retention at 100%experimental retention at 100% MeCN on a PLRP-SMeCN on a PLRP S , and the extrapolated retention at 100%column, and the extrapolated retention at 100% MeCNMeCN d from the linear region measured atobtained from the linear region measured at 0% MeCN50/60/70% MeCN. Positive values indicate extra. Positive values indicate extra on in the nonretention in the non-polar limit, consistent withpolar limit, consistent with masking/folding (i.e. chameleonicity).polarity masking/folding (i.e. chameleonicity).
d features:Method features: based method, using PLRPHPLC-based method, using PLRP-S columnS column rd the retention time (RT) of compound inRecord the retention time (RT) of compound in ring ratios ofdiffering ratios of water:acetonitrilewater:acetonitrile r regression to fit of Log k' PLRPLinear regression to fit of Log k' PLRP-S and the %S and the % nitrile; Chameleonic properties are observedacetonitrile; Chameleonic properties are observed teeper downward slope with an upas a steeper downward slope with an up-ticktick polate to calculate at 100% acetonitrile andExtrapolate to calculate at 100% acetonitrile and late Chamelogkcalculate Chamelogk using the formula:using the formula: melogk = Exp. log k' 100Chamelogk = Exp. log k' 100 - Ext. log k' 100Ext. log k' 100
This assay represents aThis assay represents asignificant step forwardsignificant step forwardin assessing molecules such asin assessing molecules such as PROTACs and optimization of their properties [7] Enabling:PROTACs and optimization of their properties [7] Enabling:
Hetero bifunctionals
Optimization to fit with target biology; e.g. crossingOptimization to fit with target biology; e.g. crossing
Heterothe BBB or efficiently accessingthe BBB or efficiently accessingintracellular targetsintracellular targetsPeptides bifunctionals
Characterization of molecules to guideCharacterization of molecules to guidedesign and design and create a robust pre-clinical data packagecreate a robust pre-clinical data package Understand theGlues
Understand therelationship between biophysicalrelationship between biophysicalMacrocycles and cell-basedand cell-basedresults results
Peptides
A key part of your drug discovery programA key part of your drug discovery program o2h offers the o2h offers the ChameLogK assay as partChameLogK assay as part of a comprehensive suite of ADMET andof a comprehensive suite of ADMET and assay biology, eassay biology, enabling fullnabling full characterisation of compoundscharacterisation of compounds
Graphics adapted from [2] and [5]Graphics adapted from [2] and [5]
o2h also offers a range of integratedo2h also offers a range of integrated services linking directly to this field:services linking directly to this field:
ReferencesReferences l (2021) ‘Prediction of Chameleonic Efficiency’,[1] David et al (2021) ‘Prediction of Chameleonic Efficiency’,ChemMedChem 16(7), pp2669ChemMedChem 16(7), pp2669-26852685 et al, (2020) ‘[2] Danielas et al, (2020) ‘Solution Conformations Explain the ChameleonicSolution Conformations Explain the Chameleonic BehaviourBehaviour of Macrocyclic Drugs’,of Macrocyclic Drugs’,Nature 26(23), pp5231Nature5244 26(23), pp5231-5244 nd Lokey, (2017) ‘Chapter 5:[3] Bockus and Lokey, (2017) ‘Chapter 5:Bioactive and MembraneBioactive and Membrane-Permeable Cyclic Peptide Natural Products’,Permeable Cyclic Peptide Natural Products’,Practical Medicinal Practical Medicinal ith Macrocycles: Design, Synthesis, and Case Studies,Chemistry with Macrocycles: Design, Synthesis, and Case Studies,John Wiley and Sons, Inc.John Wiley and Sons, Inc. et al, (2023), ‘Cell[4] Ramelotpermeable chameleonic peptides: Exploiting conformational dynamics in de novo cyclic peptide design,et al, (2023), ‘Cell-permeable chameleonic peptides: Exploiting conformational dynamics in de novo cyclic peptide design, ion in Structural Biology 80, p102603Current Opinion in Structural Biology 80, p102603 enez et al (2023), ‘[5] Garcia Jimenez et al (2023), ‘Chamelogk: A ChromatographicChamelogk: A Chromatographic Chameleonicity Quantifier to Design Orally Bioavailable Beyond-Rule-Chameleonicity Quantifier to Design Orally Bioavailable Beyond Rule ournal of Medicinal Chemistry 66(15), p10681.of-5 Drugs’, Journal of Medicinal Chemistry 66(15), p10681.Seminal article by Giulia Caron’s groupSeminal article by Giulia Caron’s group al (2014), ‘EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides’,[6] Goetz et al (2014), ‘EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides’, al Chemistry Letters, 5(10).ACS Medicinal Chemistry Letters, 5(10). t al (2024), ‘Exploring the chemical space of orally bioavailable PROTACs’, Drug Discovery Today 29(4), p103917.[7] Apprato et al (2024), ‘Exploring the chemical space of orally bioavailable PROTACs’, Drug Discovery Today 29(4), p103917.
PROTACs: off PROTACs:the shelf toolbox, featuring >100 buildingoff-the-shelf-toolbox, featuring >100 building blocks, a range of E3 ligands and readyblocks, a range of E3 ligands and ready-to-use linkersto use linkers Custom Peptide SynthesisCustom Peptide Synthesis: parallel and solid & solution: parallel and solid & solution phase; ‘peptide guarantee’phase; ‘peptide guarantee’ Macrocycles and natural product chemistryMacrocycles and natural product chemistry Molecular gluesMolecular glues, with comprehensive assay suite for in, with comprehensive assay suite for incell target engagement and degradationcell target engagement and degradation Medicinal and syntheticMedicinal and syntheticchemistry chemistry nt to learn more about this assayWant to learn more about this assay
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