VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells.

Directed evolution, artificial selection toward designed objectives, is routinely used to develop new molecular tools and therapeutics. Successful directed molecular evolution campaigns repeatedly test diverse sequences with a designed selective pressure. Unicellular organisms and their viral pathogens are exceptional for this purpose and have been used for decades. However, many desirable targets of directed evolution perform poorly or unnaturally in unicellular backgrounds. Here, we present a system for facile directed evolution in mammalian cells. Using the RNA alphavirus Sindbis as a vector for heredity and diversity, we achieved 24-h selection cycles surpassing 10-3 mutations per base. Selection is achieved through genetically actuated sequences internal to the host cell, thus the system's name: viral evolution of genetically actuating sequences, or "VEGAS." Using VEGAS, we evolve transcription factors, GPCRs, and allosteric nanobodies toward functional signaling endpoints each in less than 1 weeks' time.

Genetically-encoded BRET probes shed light on ligand bias-induced variable ion selectivity in TRPV1 and P2X5/7.

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Pharmacological characterization of allosteric modulators: A case for chemokine receptors.

Chemokine receptors are relevant targets for a multitude of immunological diseases, but drug attrition for these receptors is remarkably high. While many drug discovery programs have been pursued, most prospective drugs failed in the follow-up studies due to clinical inefficacy, and hence there is a clear need for alternative approaches. Allosteric modulators of receptor function represent an excellent opportunity for novel drugs, as they modulate receptor activation in a controlled manner and display increased selectivity, and their pharmacological profile can be insurmountable. Here, we discuss allosteric ligands and their pharmacological characterization for modulation of chemokine receptors. Ligands are included if (1) they show clear signs of allosteric modulation in vitro and (2) display evidence of binding in a topologically distinct manner compared to endogenous chemokines. We discuss how allosteric ligands affect binding of orthosteric (endogenous) ligands in terms of affinity as well as binding kinetics in radioligand binding assays. Moreover, their effects on signaling events in functional assays and how their binding site can be elucidated are specified. We substantiate this with examples of published allosteric ligands targeting chemokine receptors and hypothetical graphs of pharmacological behavior. This review should serve as an effective starting point for setting up assays for characterizing allosteric ligands to develop safer and more efficacious drugs for chemokine receptors and, ultimately, other G protein-coupled receptors.

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action.

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.

A novel machine learning-based screening identifies statins as inhibitors of the calcium pump SERCA.

We report a novel small-molecule screening approach that combines data augmentation and machine learning to identify Food and Drug Administration (FDA)-approved drugs interacting with the calcium pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA) from skeletal (SERCA1a) and cardiac (SERCA2a) muscle. This approach uses information about small-molecule effectors to map and probe the chemical space of pharmacological targets, thus allowing to screen with high precision large databases of small molecules, including approved and investigational drugs. We chose SERCA because it plays a major role in the excitation-contraction-relaxation cycle in muscle and it represents a major target in both skeletal and cardiac muscle. The machine learning model predicted that SERCA1a and SERCA2a are pharmacological targets for seven statins, a group of FDA-approved 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used in the clinic as lipid-lowering medications. We validated the machine learning predictions by using in vitro ATPase assays to show that several FDA-approved statins are partial inhibitors of SERCA1a and SERCA2a. Complementary atomistic simulations predict that these drugs bind to two different allosteric sites of the pump. Our findings suggest that SERCA-mediated Ca2+ transport may be targeted by some statins (e.g., atorvastatin), thus providing a molecular pathway to explain statin-associated toxicity reported in the literature. These studies show the applicability of data augmentation and machine learning-based screening as a general platform for the identification of off-target interactions and the applicability of this approach extends to drug discovery.

The molecular basis of dapsone activation of CYP2C9-catalyzed nonsteroidal anti-inflammatory drug oxidation.

Positive heterotropic cooperativity, or "activation," results in an instantaneous increase in enzyme activity in the absence of an increase in protein expression. Thus, cytochrome P450 (CYP) enzyme activation presents as a potential drug-drug interaction mechanism. It has been demonstrated previously that dapsone activates the CYP2C9-catalyzed oxidation of a number of nonsteroidal anti-inflammatory drugs in vitro. Here, we conducted molecular dynamics simulations (MDS) together with enzyme kinetic investigations and site-directed mutagenesis to elucidate the molecular basis of the activation of CYP2C9-catalyzed S-flurbiprofen 4'-hydroxylation and S-naproxen O-demethylation by dapsone. Supplementation of incubations of recombinant CYP2C9 with dapsone increased the catalytic efficiency of flurbiprofen and naproxen oxidation by 2.3- and 16.5-fold, respectively. MDS demonstrated that activation arises predominantly from aromatic interactions between the substrate, dapsone, and the phenyl rings of Phe114 and Phe476 within a common binding domain of the CYP2C9 active site, rather than involvement of a distinct effector site. Mutagenesis of Phe114 and Phe476 abrogated flurbiprofen and naproxen oxidation, and MDS and kinetic studies with the CYP2C9 mutants further identified a pivotal role of Phe476 in dapsone activation. MDS additionally showed that aromatic stacking interactions between two molecules of naproxen are necessary for binding in a catalytically favorable orientation. In contrast to flurbiprofen and naproxen, dapsone did not activate the 4'-hydroxylation of diclofenac, suggesting that the CYP2C9 active site favors cooperative binding of nonsteroidal anti-inflammatory drugs with a planar or near-planar geometry. More generally, the work confirms the utility of MDS for investigating ligand binding in CYP enzymes.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

[Study of antitumor effects of human placenta hydrolysate on PC-3, OAW-42, BT-474 cell cultures].

AIM: To investigate the antitumor effects of human placenta hydrolysate (HPH) peptides on three hormone-dependent human cell lines: prostate adenocarcinoma, breast carcinoma, and ovarian cancer by metabolic analysis of cell cultures. MATERIALS AND METHODS: The effect of HPH on tumor and control tumor cell lines was evaluated. Study stages: (A) de novo peptide sequencing by collision-induced dissociation mass spectrometry; (B) detection of peptides with anti-tumor properties; (C) expert analysis of the obtained lists of peptides. RESULTS: Dose-dependent cytotoxic effects of HPH on three tumor cell lines are shown: PC-3 (human prostate adenocarcinomas), OAW-42 (human ovarian cancer), BT-474 (human breast carcinomas), and IC50 constants (1.3-2.8 mg/ml) were obtained. The analysis of the HPH peptide fraction showed more than 70 peptides with antitumor properties in the composition of this HPH, including kinase inhibitors: mitogen-activated protein kinases, kappa-bi nuclear factor inhibitor kinase, AKT serine/threonine kinase 1, protein kinase C zeta, interleukin-1 receptor-associated kinase 4 and cyclin-dependent kinase 1. CONCLUSION: The results of the study indicate not only the oncological safety of the HPH used in therapy but also the mild antitumor effects of this HPH at high concentrations.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Facilitation of hERG Activation by Its Blocker: A Mechanism to Reduce Drug-Induced Proarrhythmic Risk.

Modulation of the human Ether-à-go-go-Related Gene (hERG) channel, a crucial voltage-gated potassium channel in the repolarization of action potentials in ventricular myocytes of the heart, has significant implications on cardiac electrophysiology and can be either antiarrhythmic or proarrhythmic. For example, hERG channel blockade is a leading cause of long QT syndrome and potentially life-threatening arrhythmias, such as torsades de pointes. Conversely, hERG channel blockade is the mechanism of action of Class III antiarrhythmic agents in terminating ventricular tachycardia and fibrillation. In recent years, it has been recognized that less proarrhythmic hERG blockers with clinical potential or Class III antiarrhythmic agents exhibit, in addition to their hERG-blocking activity, a second action that facilitates the voltage-dependent activation of the hERG channel. This facilitation is believed to reduce the proarrhythmic potential by supporting the final repolarizing of action potentials. This review covers the pharmacological characteristics of hERG blockers/facilitators, the molecular mechanisms underlying facilitation, and their clinical significance, as well as unresolved issues and requirements for research in the fields of ion channel pharmacology and drug-induced arrhythmias.

A general method for quantifying ligand binding to unmodified receptors using Gaussia luciferase.

Reliable measurement of ligand binding to cell surface receptors is of outstanding biological and pharmacological importance. Resonance energy transfer-based assays are powerful approaches to achieve this goal, but the currently available methods are hindered by the necessity of receptor tagging, which can potentially alter ligand binding properties. Therefore, we developed a tag-free system to measure ligand‒receptor interactions in live cells using the Gaussia luciferase (GLuc) as a bioluminescence resonance energy transfer donor. GLuc is as small as the commonly applied Nanoluciferase but has enhanced brightness, and its proper substrate is the frequently used coelenterazine. In our assay, bystander bioluminescence resonance energy transfer is detected between a GLuc-based extracellular surface biosensor and fluorescent ligands bound to their unmodified receptors. The broad spectrum of applications includes equilibrium and kinetic ligand binding measurements for both labeled and competitive unlabeled ligands, and the assay can be utilized for different classes of plasma membrane receptors. Furthermore, the assay is suitable for high-throughput screening, as evidenced by the identification of novel α1 adrenergic receptor ligands. Our data demonstrate that GLuc-based biosensors provide a simple, sensitive, and cost-efficient platform for drug characterization and development.

Can be marine bioactive peptides (MBAs) lead the future of foodomics for human health?

Marine organisms are considered a cache of biologically active metabolites with pharmaceutical, functional, and nutraceutical properties. Among these, marine bioactive peptides (MBAs) present in diverse marine species (fish, sponges, cyanobacteria, fungi, ascidians, seaweeds, & mollusks) have acquired attention owing to their broad-spectrum health-promoting benefits. Nowadays, scientists are keener exploring marine bioactive peptides precisely due to their unique structural and biological properties. These MBAs have reported ameliorating potential against different diseases like hypertension, diabetes, obesity, HIV, cancer, oxidation, and inflammation. Furthermore, MBAs isolated from various marine organisms may also have a beneficial role in the cosmetic, nutraceutical, and food industries. Few marine peptides and their derivative are approved for commercial use, while many MBAs are in various pre-clinical and clinical trials. This review mainly focuses on the diversity of marine bioactive peptides in marine organisms and their production procedures, such as chemical and enzymatic hydrolysis. Moreover, MBAs' therapeutic and biological potential has also been critically discussed herein, along with their status in drug discovery, pre-clinical and clinical trials.

Alpelisib to treat CLOVES syndrome, a member of the PIK3CA-related overgrowth syndrome spectrum.

CLOVES syndrome is a rare congenital overgrowth disorder caused by mutations in the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) gene. It is part of the PIK3CA-related overgrowth syndrome (PROS) spectrum and its treatment is challenging. PROS malformations have traditionally been treated by surgery, but research into pharmacological treatments capable of blocking the PIK/AKT/mTOR pathway has increased over the past decade. The results have been promising and suggest that compassionate use of these treatments in patients with PROS disorders could have clinical benefits. Another promising drug is alpelisib (BYL719), which is a selective inhibitor that competitively binds to the p110a subunit of PIK3 in the intracellular PI3K/AKT signalling pathway. Compassionate use of low-dose alpelisib had striking effects in an uncontrolled case series of 19 PROS patients, several with life-threatening complications. Moreover, there were few adverse effects and the treatment did not impair linear growth, despite the young age of many of the patients. We present the case of a patient with CLOVES syndrome who was started on compassionate treatment with alpelisib after surgical debulking of a cystic lymphangioma and treatment with sirolimus. This promising drug significantly reduced the size of the lymphangioma and prevented progression of the tissue overgrowth in the gluteal region. This case suggests that low-dose PI3K inhibition may provide collateral benefits that extend beyond mitigation of disease-specific features of PROS.

Comparison of the Chemical Components, Efficacy and Mechanisms of Action of Chrysanthemum morifolium Flower and Its Wild Relative Chrysanthemum indicum Flower against Liver-Fire Hyperactivity Syndrome of Hypertension via Integrative Analyses.

To clarify the differences in the clinical application scope of Chrysanthemum morifolium flower (CMF) and Chrysanthemum indicum flower (CIF), two herbs of similar origin, an integrated strategy of network pharmacology, molecular pharmacology, and metabolomics was employed, with a view to investigating the commonalities and dissimilarities in chemical components, efficacy and mechanisms of action. Initial HPLC-Q-TOF-MS analysis revealed that CMF and CIF had different flavonoid constituents. The biological processes underlying the therapeutic effects of CMF and CIF on liver-fire hyperactivity syndrome of hypertension (LFHSH) were predicted to be related to inflammatory response, fatty acid production, and other pathways based on network pharmacology analysis. ELISA, molecular docking, Western blot, and metabolomics techniques showed similar effects of CMF and CIF in lowering blood pressure, resistance to tissue, organ and functional damage, and dyslipidemia. However, distinct effects were found in the regulation of inflammatory response, PI3K-Akt and NF-κB signaling pathways, lipid anabolism, renin-angiotensin system, and metabolic abnormalities. The comparable efficacies of CMF and CIF, despite having distinct mechanisms of action, may be attributed to the integration and counteraction of their different regulating capabilities on the above anti-LFHSH mechanisms. This study offers a vital platform for assessment of differential and precise applications of herbs of close origin with similar but slightly different medicinal properties, and provides a research strategy for bridging Chinese medicine and modern precision medicine.

Exploitation of dihydroorotate dehydrogenase (DHODH) and p53 activation as therapeutic targets: A case study in polypharmacology.

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.

Delineation of molecular determinants for FR900359 inhibition of Gq/11 unlocks inhibition of Gαs.

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11 subfamily consists of Gq, G11, G14, and G16 proteins, of which all but G16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families, Gs, Gi/o, and G12/13, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits Gq/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359-binding site in Gq and Gs We found that the activity of a Gs mutant with a Gq-like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in Gs In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive Gs, Gi/o, and G12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved, opening up the possibility of targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359-sensitive Gαs mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.

Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein-coupled receptor (CLR), which heterodimerizes with three receptor activity-modifying proteins (RAMP1-3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein-tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

A suite of mathematical solutions to describe ternary complex formation and their application to targeted protein degradation by heterobifunctional ligands.

Small molecule-induced targeted protein degradation by heterobifunctional ligands or molecular glues represents a new modality in drug development, allowing development of therapeutic agents for targets previously considered undruggable. Successful target engagement requires the formation of a ternary complex (TC) when the ligand brings its target protein in contact with an E3 ubiquitin ligase. Unlike traditional drugs, where target engagement can be described by a simple bimolecular equilibrium equation, similar mathematical tools are currently not available to describe TC formation in a universal manner. This current limitation substantially increases the challenges of developing drugs with targeted protein degradation mechanism. In this article, I provide a full, exact, and universal mathematical description of the TC system at equilibrium for the first time. I have also constructed a comprehensive suite of mathematical tools for quantitative measurement of target engagement and equilibrium constants from experimental data. Mechanistic explanations are provided for many common challenges associated with developing this type of therapeutic agent. Insights from these analyses provide testable hypotheses and grant direction to drug development efforts in this promising area. The mathematical and analytical tools described in this article may also have broader applications in other areas of biology and chemistry in which ternary complexes are observed.

Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR-one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)-have provided significant health benefits to CF patients. Here, we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein's proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.

The African natural product knipholone anthrone and its analogue anthralin (dithranol) enhance HIV-1 latency reversal.

A sterilizing or functional cure for HIV is currently precluded by resting CD4+ T cells that harbor latent but replication-competent provirus. The "shock-and-kill" pharmacological ap-proach aims to reactivate provirus expression in the presence of antiretroviral therapy and target virus-expressing cells for elimination. However, no latency reversal agent (LRA) to date effectively clears viral reservoirs in humans, suggesting a need for new LRAs and LRA combinations. Here, we screened 216 compounds from the pan-African Natural Product Library and identified knipholone anthrone (KA) and its basic building block anthralin (dithranol) as novel LRAs that reverse viral latency at low micromolar concentrations in multiple cell lines. Neither agent's activity depends on protein kinase C; nor do they inhibit class I/II histone deacetylases. However, they are differentially modulated by oxidative stress and metal ions and induce distinct patterns of global gene expression from established LRAs. When applied in combination, both KA and anthralin synergize with LRAs representing multiple functional classes. Finally, KA induces both HIV RNA and protein in primary cells from HIV-infected donors. Taken together, we describe two novel LRAs that enhance the activities of multiple "shock-and-kill" agents, which in turn may inform ongoing LRA combination therapy efforts.