Methoxyfuranocoumarins of Natural Origin-Updating Biological Activity Research and Searching for New Directions-A Review.

Plant secondary metabolites, including furanocoumarins, have attracted attention for decades as active molecules with therapeutic potential, especially those occurring in a limited number of species as evolutionarily specific and chemotaxonomically important. The most famous methoxyfuranocoumarins (MFCs), bergapten, xanthotoxin, isopimpinellin, phellopterin, byakangelicol, byakangelicin, isobergapten, pimpinellin, sphondin, as well as rare ones such as peucedanin and 8-methoxypeucedanin, apaensin, cnidilin, moellendorffiline and dahuribiethrins, have recently been investigated for their various biological activities. The α-glucosidase inhibitory activity and antioxidant potential of moellendorffiline, the antiproliferative and proapoptotic properties of non-UV-activated bergapten and xanthotoxin, the effect of MFC on the activity of tyrosinase, acetyl- and butylcholinesterase, and the role of these compounds as adjuvants in anticancer and antibacterial tests have been confirmed. The anticonvulsant effects of halfordin, the antidepressant effects of xanthotoxin, and the antiadipogenic, neuroprotective, anti-amyloid-ß, and anti-inflammatory (via increasing SIRT 1 protein expression) properties of phellopterin, as well as the activity of sphondin against hepatitis B virus, have also attracted interest. It is worth paying attention to the agonistic effect of xanthotoxin on bitter taste receptors (TAS2Rs) on cardiomyocytes, which may be important in the future treatment of tachycardia, as well as the significant anti-inflammatory activity of dahuribiethrins. It should be emphasized that MFCs, although in many cases isolated for the first time many years ago, are still of great interest as bioactive molecules. The aim of this review is to highlight key recent developments in the study of the diverse biological activities of MFCs and attempt to highlight promising directions for their further research. Where possible, descriptions of the mechanisms of action of MFC are provided, which is related to the constantly discovered therapeutic potential of these molecules. The review covers the results of experiments from the last ten years (2014-2023) conducted on isolated natural cMFCs and includes the activity of molecules that have not been activated by UV rays.

Novel N-Acylethanolamide Derivatives Affect Body Weight and Energy Balance.

Introduction - The obesity pandemic is multifactorial. Nutritional, pharmacologic and surgical interventions are limited in reach and efficacy, raising need for new therapeutics. Aims - Characterization of anorexigenic and cognitive effect and central mechanism of action of novel N-acylethanolamide derivatives. Methods - Sabra mice divided to similar experimental groups, injected IP with: oleyl-L-leucinolamide (1 A), linoleyl-L-leucinolamide (4 A), linoleyl-L-valinolamide (5 A), oleyl-oxycarbonyl-L-valinolamide (1 B), oleyl-oxycarbonyl-D-valinolamide (2 B), oleylamine-carbonyl-L-valinolamide (3 B), oleylamine-carbonyl-D-valinolamide (4 B), and oleyl-L-hydroxyvalineamide (5 B). Control group with vehicle. Body weight and food consumption followed for 39 days. Motor activity and cognitive function by open field test and eight-arm maze. Mice sacrificed and mechanism of action investigated by qPCR. The genes analyzed involved in energy balance and regulation of appetite. Catecholamines and serotonin evaluated. Results - Compounds 1 A, 5 A, 1 B-4 B, caused significant weight loss of 4.2-5.6 % and 5 A, 1 B-4 B, improved cognitive function following 8 i. p. injections of 1 mg/kg during 39 days, by different mechanisms. 5 A, 3 B and 4 B decreased food consumption, whereas 1 A, 5 A and 2 B increased motor activity. 1 A, 4 A, 1 B and 3 B elevated SIRT-1, associated with survival. POMC upregulated by 1 B and 2 B, CART by 1 B, 2 B and 1 A. NPY and CAMKK2 downregulated by 5 A. 4 B enhanced 5-HT levels. 4 A, 5 A, 1 B, 4 B, 5 B decreased FAAH, showing long lasting effect. Conclusions - These new compounds might be developed for the treatment of obesity and for improved cognitive function.

Rational Design of a Novel Tubulin Inhibitor with a Unique Mechanism of Action.

In this study, we capitalized on our previously performed crystallographic fragment screen and developed the antitubulin small molecule Todalam with only two rounds of straightforward chemical synthesis. Todalam binds to a novel tubulin site, disrupts microtubule networks in cells, arrests cells in G2/M, induces cell death, and synergizes with vinblastine. The compound destabilizes microtubules by acting as a molecular plug that sterically inhibits the curved-to-straight conformational switch in the α-tubulin subunit, and by sequestering tubulin dimers into assembly incompetent oligomers. Our results describe for the first time the generation of a fully rationally designed small molecule tubulin inhibitor from a fragment, which displays a unique molecular mechanism of action. They thus demonstrate the usefulness of tubulin-binding fragments as valuable starting points for innovative antitubulin drug and chemical probe discovery campaigns.

Recent advances in proteome-wide label-free target deconvolution for bioactive small molecules.

Small-molecule drugs modulate biological processes and disease states through engagement of target proteins in cells. Assessing drug-target engagement on a proteome-wide scale is of utmost importance in better understanding the molecular mechanisms of action of observed beneficial and adverse effects, as well as in developing next generation tool compounds and drugs with better efficacies and specificities. However, systematic assessment of drug-target engagement has been an arduous task. With the continuous development of mass spectrometry-based proteomics instruments and techniques, various chemical proteomics approaches for drug target deconvolution (i.e., the identification of molecular target for drugs) have emerged. Among these, the label-free target deconvolution approaches that do not involve the chemical modification of compounds of interest, have gained increased attention in the community. Here we provide an overview of the basic principles and recent biological applications of the most important label-free methods including the cellular thermal shift assay, pulse proteolysis, chemical denaturant and protein precipitation, stability of proteins from rates of oxidation, drug affinity responsive target stability, limited proteolysis, and solvent-induced protein precipitation. The state-of-the-art technical implications and future outlook for the label-free approaches are also discussed.

Thyroid hormone and thyroid hormone nuclear receptors: History and present state of art.

The present review traces the road leading to discovery of L-thyroxine, thyroid hormone (3,5,3´-triiodo-L-thyronine, T3) and its cognate nuclear receptors. Thyroid hormone is a pleio-tropic regulator of growth, differentiation, and tissue homeostasis in higher organisms. The major site of the thyroid hormone action is predominantly a cell nucleus. T3 specific binding sites in the cell nuclei have opened a new era in the field of the thyroid hormone receptors (TRs) discovery. T3 actions are mediated by high affinity nuclear TRs, TRalpha and TRbeta, which function as T3-activated transcription factors playing an essential role as transcription-modulating proteins affecting the transcriptional responses in target genes. Discovery and characterization of nuclear retinoid X receptors (RXRs), which form with TRs a heterodimer RXR/TR, positioned RXRs at the epicenter of molecular endocrinology. Transcriptional control via nuclear RXR/TR heterodimer represents a direct action of thyroid hormone. T3 plays a crucial role in the development of brain, it exerts significant effects on the cardiovascular system, skeletal muscle contractile function, bone development and growth, both female and male reproductive systems, and skin. It plays an important role in maintaining the hepatic, kidney and intestine homeostasis and in pancreas, it stimulates the beta-cell proliferation and survival. The TRs cross-talk with other signaling pathways intensifies the T3 action at cellular level. The role of thyroid hormone in human cancers, acting via its cognate nuclear receptors, has not been fully elucidated yet. This review is aimed to describe the history of T3 receptors, starting from discovery of T3 binding sites in the cell nuclei to revelation of T3 receptors as T3-inducible transcription factors in relation to T3 action at cellular level. It also focuses on milestones of investigation, comprising RXR/TR dimerization, cross-talk between T3 receptors, and other regulatory pathways within the cell and mainly on genomic action of T3. This review also focuses on novel directions of investigation on relationships between T3 receptors and cancer. Based on the update of available literature and the author's experimental experience, it is devoted to clinicians and medical students.

Effect of Sepatronium Bromide (YM-155) on DNA Double-Strand Breaks Repair in Cancer Cells.

Survivin, as an antiapoptotic protein often overexpressed in cancer cells, is a logical target for potential cancer treatment. By overexpressing survivin, cancer cells can avoid apoptotic cell death and often become resistant to treatments, representing a significant obstacle in modern oncology. A survivin suppressor, an imidazolium-based compound known as YM-155, is nowadays studied as an attractive anticancer agent. Although survivin suppression by YM-155 is evident, researchers started to report that YM-155 is also an inducer of DNA damage introducing yet another anticancer mechanism of this drug. Moreover, the concentrations of YM-155 for DNA damage induction seems to be far lower than those needed for survivin inhibition. Understanding the molecular mechanism of action of YM-155 is of vital importance for modern personalized medicine involving the selection of responsive patients and possible treatment combinations. This review focuses mainly on the documented effects of YM-155 on DNA damage signaling pathways. It summarizes up to date literature, and it outlines the molecular mechanism of YM-155 action in the context of the DNA damage field.

Molecular Mechanism of Action of RORγt Agonists and Inverse Agonists: Insights from Molecular Dynamics Simulation.

As an attractive drug-target, retinoic acid receptor-related orphan receptor-gamma-t (RORγt) has been employed widely to develop clinically relevant small molecular modulators as potent therapy for autoimmune disease and cancer, but its molecular mechanism of action (MOA) remains unclear. In the present study, we designed and discovered two novel RORγt ligands that are similar in structure, but different in efficacy. Using fluorescence resonance energy transfer (FRET) assay, compound 1 was identified as an agonist with an EC50 of 3.7 µM (max. act.: 78%), while compound 2 as an inverse agonist with an IC50 value of 2.0 µM (max. inh.: 61%). We performed molecular dynamics (MD) simulations, and elucidated the MOA of RORγt agonist and inverse agonist. Through the analyses of our MD results, we found that, after RORγt is bound with the agonist 1, the side chain of Trp317 stays in the gauche- conformation, and thus helps to form the hydrogen bond, His479-Trp502, and a large hydrophobic network among H11, H11', and H12. All these interactions stabilize the H12, and helps the receptor recruit the coactivator. When the RORγt is bound with the inverse agonist 2, the side chain of Trp317 is forced to adopt the trans conformation, and these presumed interactions are partially destroyed. Taken together, the critical role of residue Trp317 could be viewed as the driving force for the activation of RORγt.

Discovery of a novel prolyl-tRNA synthetase inhibitor and elucidation of its binding mode to the ATP site in complex with l-proline.

Prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase family of enzymes and catalyzes the synthesis of prolyl-tRNAPro using ATP, l-proline, and tRNAPro as substrates. An ATP-dependent PRS inhibitor, halofuginone, was shown to suppress autoimmune responses, suggesting that the inhibition of PRS is a potential therapeutic approach for inflammatory diseases. Although a few PRS inhibitors have been derivatized from natural sources or substrate mimetics, small-molecule human PRS inhibitors have not been reported. In this study, we discovered a novel series of pyrazinamide PRS inhibitors from a compound library using pre-transfer editing activity of human PRS enzyme. Steady-state biochemical analysis on the inhibitory mode revealed its distinctive characteristics of inhibition with proline uncompetition and ATP competition. The binding activity of a representative compound was time-dependently potentiated by the presence of l-proline with Kd of 0.76 nM. Thermal shift assays demonstrated the stabilization of PRS in complex with l-proline and pyrazinamide PRS inhibitors. The binding mode of the PRS inhibitor to the ATP site of PRS enzyme was elucidated using the ternary complex crystal structure with l-proline. The results demonstrated the different inhibitory and binding mode of pyrazinamide PRS inhibitors from preceding halofuginone. Furthermore, the PRS inhibitor inhibited intracellular protein synthesis via a different mode than halofuginone. In conclusion, we have identified a novel drug-like PRS inhibitor with a distinctive binding mode. This inhibitor was effective in a cellular context. Thus, the series of PRS inhibitors are considered to be applicable to further development with differentiation from preceding halofuginone.

Structural Basis of Microtubule Stabilization by Discodermolide.

Microtubule-stabilizing agents (MSAs) are widely used in chemotherapy. Using X-ray crystallography we elucidated the detailed binding modes of two potent MSAs, (+)-discodermolide (DDM) and the DDM-paclitaxel hybrid KS-1-199-32, in the taxane pocket of ß-tubulin. The two compounds bind in a very similar hairpin conformation, as previously observed in solution. However, they stabilize the M-loop of ß-tubulin differently: KS-1-199-32 induces an M-loop helical conformation that is not observed for DDM. In the context of the microtubule structure, both MSAs connect the ß-tubulin helices H6 and H7 and loop S9-S10 with the M-loop. This is similar to the structural effects elicited by epothilone A, but distinct from paclitaxel. Together, our data reveal differential binding mechanisms of DDM and KS-1-199-32 on tubulin.

Metal-Catalyzed Oxidation and Photo-oxidation of Glucagon.

The oxidation of glucagon by the H2O2/Cu(2+) system and by simulated sunlight was studied using HPLC-MS methodologies. It was found that copper ion-catalyzed oxidation is much faster in the residue 1-12 region than in photo-oxidation, but it is slower than photo-oxidation in the residue 18-29 region. This difference is due to the unique feature of the primary sequence of glucagon. The residue 1-12 region contains His-1 and Asp-9 that can bind to Cu(2+) ions and catalyze the oxidation of His-1 and Tyr-10, while the residue 18-29 region lacks these charged residues near the liable Met-27 and Trp-25 and hence no catalysis by the neighboring groups occurs. Fragment (residue 13-17) was more stable than the other regions of the peptide toward photo-oxidation because it contains only one oxidizable residue, Tyr-13. These findings may help explain the mechanism of action of glucagon and provide some hints for the development of effective anti-diabetic drug molecules and stable glucagon formulations.

Structural basis of microtubule stabilization by laulimalide and peloruside A.

Laulimalide and peloruside A are microtubule-stabilizing agents (MSAs), the mechanism of action on microtubules of which is poorly defined. Here, using X-ray crystallography it is shown that laulimalide and peloruside A bind to a unique non-taxane site on ß-tubulin and use their respective macrolide core structures to interact with a second tubulin dimer across protofilaments. At the same time, they allosterically stabilize the taxane-site M-loop that establishes lateral tubulin contacts in microtubules. Structures of ternary complexes of tubulin with laulimalide/peloruside A and epothilone A are also solved, and a crosstalk between the laulimalide/peloruside and taxane sites via the M-loop of ß-tubulin is found. Together, the data define the mechanism of action of laulimalide and peloruside A on tubulin and microtubules. The data further provide a structural framework for understanding the synergy observed between two classes of MSAs in tubulin assembly and the inhibition of cancer cell growth.

Targeting pan-essential pathways in cancer with cytotoxic chemotherapy: challenges and opportunities.

Cytotoxic chemotherapy remains a key modality in cancer treatment. These therapies, successfully used for decades, continue to transform the lives of cancer patients daily. With the high attrition rate of current oncology drug development, combined with the knowledge that most new therapies do not displace standard-of-care treatments and that many healthcare systems cannot afford these new therapies; cytotoxic chemotherapies will remain an important component of cancer therapy for many years to come. The clinical value of these therapies is often under-appreciated within the pre-clinical cancer research community, where this diverse class of agents are often grouped together as non-specific cellular poisons killing tumor cells based solely upon proliferation rate; however, this is inaccurate. This review article seeks to reaffirm the importance of focusing research efforts upon improving our basic understanding of how these drugs work, discussing their ability to target pan-essential pathways in cancer cells, the relationship of this to the chemotherapeutic window, and highlighting basic science approaches that can be employed towards refining their use.

Study of the Active Components and Molecular Mechanism of Tripterygium wilfordii in the Treatment of Diabetic Nephropathy.

We aimed to explore the active ingredients and molecular mechanism of Tripterygium wilfordii (TW) in the treatment of diabetic nephropathy (DN) through network pharmacology and molecular biology. First, the active ingredients and potential targets of TW were obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and related literature materials, and Cytoscape 3.7.2 software was used to construct the active ingredient-target network diagram of TW. Second, the target set of DN was obtained through the disease database, and the potential targets of TW in the treatment of DN were screened through a Venn diagram. A protein interaction network diagram (PPI) was constructed with the help of the String platform and Cytoscape 3.7.2. Third, the ClueGO plug-in tool was used to enrich the GO biological process and the KEGG metabolic pathway. Finally, molecular docking experiments and cell pathway analyses were performed. As a result, a total of 52 active ingredients of TW were screened, and 141 predicted targets and 49 target genes related to DN were identified. The biological process of GO is mediated mainly through the regulation of oxygen metabolism, endothelial cell proliferation, acute inflammation, apoptotic signal transduction pathway, fibroblast proliferation, positive regulation of cyclase activity, adipocyte differentiation and other biological processes. KEGG enrichment analysis showed that the main pathways involved were AGE-RAGE, vascular endothelial growth factor, HIF-1, IL-17, relaxin signalling pathway, TNF, Fc epsilon RI, insulin resistance and other signaling pathways. It can be concluded that TW may treat DN by reducing inflammation, reducing antioxidative stress, regulating immunity, improving vascular disease, reducing insulin resistance, delaying renal fibrosis, repairing podocytes, and reducing cell apoptosis, among others, with multicomponent, multitarget and multisystem characteristics.

Potent Molecular Feature-based Neutralizing Monoclonal Antibodies as Promising Therapeutics Against SARS-CoV-2 Infection.

The 2019-2020 winter was marked by the emergence of a new coronavirus (SARS-CoV-2) related disease (COVID-19), which started in Wuhan, China. Its high human-to-human transmission ability led to a worldwide spread within few weeks and has caused substantial human loss. Mechanical antiviral control approach, drug repositioning, and use of COVID-19 convalescent plasmas (CPs) were the first line strategies utilized to mitigate the viral spread, yet insufficient. The urgent need to contain this deadly pandemic has led searchers and pharmaceutical companies to develop vaccines. However, not all vaccines manufactured are safe. Besides, an alternative and effective treatment option for such an infectious disease would include pure anti-viral neutralizing monoclonal antibodies (NmAbs), which can block the virus at specific molecular targets from entering cells by inhibiting virus-cell structural complex formation, with more safety and efficiency than the CP. Indeed, there is a lot of molecular evidence about the protector effect and the use of molecular feature-based NmAbs as promising therapeutics to contain COVID-19. Thus, from the scientific publication database screening, we here retrieved antibody-related papers and summarized the repertory of characterized NmAbs against SARS-CoV-2, their molecular neutralization mechanisms, and their immunotherapeutic pros and cons. About 500 anti-SARS-CoV-2 NmAbs, characterized through competitive binding assays and neutralization efficacy, were reported at the writing time (January 2021). All NmAbs bind respectively to SARS-CoV-2 S and exhibit high molecular neutralizing effects against wild-type and/or pseudotyped virus. Overall, we defined six NmAb groups blocking SARS-CoV-2 through different molecular neutralization mechanisms, from which five potential neutralization sites on SARS-CoV-2 S protein are described. Therefore, more efforts are needed to develop NmAbs-based cocktails to mitigate COVID-19.

In vitro and in silico studies of 8(17),12E,14-labdatrien-18-oic acid in airways smooth muscle relaxation: new molecular insights about its mechanism of action.

In the field of experimental pharmacology, researchers continuously investigate new relaxant agents of the airway smooth muscle cells (ASMCs), since the pathophysiology of respiratory illnesses, such as asthma, involves hyperresponsiveness and changes in ASMC homeostasis. In this scenario, labdane-type diterpenes, like forskolin (FSK), are a class of compounds known for their relaxing action on smooth muscle cells (SMCs), being this phenomenon related to the direct activation of AC-cAMP-PKA pathway. Considering the continuous effort of our group to study the mechanism of action and prospecting for compounds isolated from natural sources, in this paper, we presented how the diterpene 8(17),12E,14-labdatrien-18-oic acid (LBD) promotes relaxant effect on ASMC, performing in vitro experiments using isolated guinea pig trachea and in silico molecular docking/dynamics simulations. In vitro experiments showed that in the presence of aminophylline, FSK and LBD had their relaxant effect potentiated (EC50 from 1.4 ± 0.2 × 10-5 M to 1.5 ± 0.3 × 10-6 M for LBD and from 2.0 ± 0.2 × 10-7 M to 6.4 ± 0.4 × 10-8 M for FSK) while in the presence of Rp-cAMPS this effect was attenuated (EC50 from 1.4 ± 0.2 × 10-5 M to 3 × 10-4 M for LBD and from 2.0 ± 0.2 × 10-7 to 3.1 ± 1.0 × 10-6 M for FSK). Additionally, in silico simulations evidenced that the lipophilic character of LBD is probably responsible for its stability on AC binding site. LBD presented two preferential orientations, where the double bonds of the isoprene moiety as well as the unique polar group (carboxylic acid) in this compound form important anchoring points. In this sense, we consider that the LBD can interact stabilizing the catalytic dimmer of AC as the FSK, although less efficiently.

Pironetin Binds Covalently to αCys316 and Perturbs a Major Loop and Helix of α-Tubulin to Inhibit Microtubule Formation.

Microtubule-targeting agents are among the most powerful drugs used in chemotherapy to treat cancer patients. Pironetin is a natural product that displays promising anticancer properties by binding to and potently inhibiting tubulin assembly into microtubules; however, its molecular mechanism of action remained obscure. Here, we solved the crystal structure of the tubulin-pironetin complex and found that the compound covalently binds to Cys316 of α-tubulin. The structure further revealed that pironetin perturbs the T7 loop and helix H8 of α-tubulin. Since both these elements are essential for establishing longitudinal tubulin contacts in microtubules, this result explains how pironetin inhibits the formation of microtubules. Together, our data define the molecular details of the pironetin binding site on α-tubulin and thus offer a promising basis for the rational design of pironetin variants with improved activity profiles. They further extend our knowledge on strategies evolved by natural products to target and perturb the microtubule cytoskeleton.

A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands.

BACKGROUND AND PURPOSE: The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. EXPERIMENTAL APPROACH: Using [(3) H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. KEY RESULTS: Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor-ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k-2 , was 1.2 × 10(-3) min(-1) . The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. CONCLUSIONS AND IMPLICATIONS: The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc-receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand.

The value of translational biomarkers to phenotypic assays.

Phenotypic assays are tools essential for drug discovery. Phenotypic assays have different types of endpoints depending on the goals; (1) empirical endpoints for basic research to understand the underlying biology that will lead to identification of translation biomarkers, (2) empirical endpoints to identify undesired effects related to toxicity of drug candidates, and (3) knowledge-based endpoints (biomarkers) for drug discovery which ideally are translational biomarkers that will be used to identify new drug candidates and their corresponding molecular mechanisms of action. The value of phenotypic assays is increased through effective alignment of phenotypic assay endpoints with the objectives of the relevant stage in the drug discovery and development cycle.

Label-free drug discovery.

Current drug discovery is dominated by label-dependent molecular approaches, which screen drugs in the context of a predefined and target-based hypothesis in vitro. Given that target-based discovery has not transformed the industry, phenotypic screen that identifies drugs based on a specific phenotype of cells, tissues, or animals has gained renewed interest. However, owing to the intrinsic complexity in drug-target interactions, there is often a significant gap between the phenotype screened and the ultimate molecular mechanism of action sought. This paper presents a label-free strategy for early drug discovery. This strategy combines label-free cell phenotypic profiling with computational approaches, and holds promise to bridge the gap by offering a kinetic and holistic representation of the functional consequences of drugs in disease relevant cells that is amenable to mechanistic deconvolution.