A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells.

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.

IDR-targeting compounds suppress HPV genome replication via disruption of phospho-BRD4 association with DNA damage response factors.

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Small molecule glucagon release inhibitors with activity in human islets.

Purpose: Type 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet ß-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. Results: Here, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 ß-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. Conclusion: Given the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.

Glucocorticoid mediated inhibition of LKB1 mutant non-small cell lung cancers.

The glucocorticoid receptor (GR) is an important anti-cancer target in lymphoid cancers but has been understudied in solid tumors like lung cancer, although glucocorticoids are often given with chemotherapy regimens to mitigate side effects. Here, we identify a dexamethasone-GR mediated anti-cancer response in a subset of aggressive non-small cell lung cancers (NSCLCs) that harbor Serine/Threonine Kinase 11 (STK11/LKB1) mutations. High tumor expression of carbamoyl phosphate synthase 1 (CPS1) was strongly linked to the presence of LKB1 mutations, was the best predictor of NSCLC dexamethasone (DEX) sensitivity (p < 10-16) but was not mechanistically involved in DEX sensitivity. Subcutaneous, orthotopic and metastatic NSCLC xenografts, biomarker-selected, STK11/LKB1 mutant patient derived xenografts, and genetically engineered mouse models with KRAS/LKB1 mutant lung adenocarcinomas all showed marked in vivo anti-tumor responses with the glucocorticoid dexamethasone as a single agent or in combination with cisplatin. Mechanistically, GR activation triggers G1/S cell cycle arrest in LKB1 mutant NSCLCs by inducing the expression of the cyclin-dependent kinase inhibitor, CDKN1C/p57(Kip2). All findings were confirmed with functional genomic experiments including CRISPR knockouts and exogenous expression. Importantly, DEX-GR mediated cell cycle arrest did not interfere with NSCLC radiotherapy, or platinum response in vitro or with platinum response in vivo. While DEX induced LKB1 mutant NSCLCs in vitro exhibit markers of cellular senescence and demonstrate impaired migration, in vivo DEX treatment of a patient derived xenograft (PDX) STK11/LKB1 mutant model resulted in expression of apoptosis markers. These findings identify a previously unknown GR mediated therapeutic vulnerability in STK11/LKB1 mutant NSCLCs caused by induction of p57(Kip2) expression with both STK11 mutation and high expression of CPS1 as precision medicine biomarkers of this vulnerability.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Fast-Killing Tyrosine Amide ((S)-SW228703) with Blood- and Liver-Stage Antimalarial Activity Associated with the Cyclic Amine Resistance Locus (PfCARL).

Current malaria treatments are threatened by drug resistance, and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) and P. falciparum acetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their targets are unknown. The tyramine of (S)-SW703 is shared with some reported PfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for the activity of (S)-SW703, suggesting differences in the mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity for multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

Identification of Glucose Transport Modulators In Vitro and Method for Their Deep Learning Neural Network Behavioral Evaluation in Glucose Transporter 1-Deficient Mice.

Metabolic flux augmentation via glucose transport activation may be desirable in glucose transporter 1 (Glut1) deficiency syndrome (G1D) and dementia, whereas suppression might prove useful in cancer. Using lung adenocarcinoma cells that predominantly express Glut1 relative to other glucose transporters, we screened 9,646 compounds for effects on the accumulation of an extracellularly applied fluorescent glucose analog. Five drugs currently prescribed for unrelated indications or preclinically characterized robustly enhanced intracellular fluorescence. Additionally identified were 37 novel activating and nine inhibitory compounds lacking previous biologic characterization. Because few glucose-related mechanistic or pharmacological studies were available for these compounds, we developed a method to quantify G1D mouse behavior to infer potential therapeutic value. To this end, we designed a five-track apparatus to record and evaluate spontaneous locomotion videos. We applied this to a G1D mouse model that replicates the ataxia and other manifestations cardinal to the human disorder. Because the first two drugs that we examined in this manner (baclofen and acetazolamide) exerted various impacts on several gait aspects, we used deep learning neural networks to more comprehensively assess drug effects. Using this method, 49 locomotor parameters differentiated G1D from control mice. Thus, we used parameter modifiability to quantify efficacy on gait. We tested this by measuring the effects of saline as control and glucose as G1D therapy. The results indicate that this in vivo approach can estimate preclinical suitability from the perspective of G1D locomotion. This justifies the use of this method to evaluate our drugs or other interventions and sort candidates for further investigation. SIGNIFICANCE STATEMENT: There are few or no activators and few clinical inhibitors of glucose transport. Using Glut1-rich cells exposed to a glucose analog, we identified, in highthroughput fashion, a series of novel modulators. Some were drugs used to modify unrelated processes and some represented large but little studied chemical compound families. To facilitate their preclinical efficacy characterization regardless of potential mechanism of action, we developed a gait testing platform for deep learning neural network analysis of drug impact on Glut1-deficient mouse locomotion.

GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology.

Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.

LIMS-Kinase provides sensitive and generalizable label-free in vitro measurement of kinase activity using mass spectrometry.

Measurements of kinase activity are important for kinase-directed drug development, analysis of inhibitor structure and function, and understanding mechanisms of drug resistance. Sensitive, accurate, and miniaturized assay methods are crucial for these investigations. Here, we describe a label-free, high-throughput mass spectrometry-based assay for studying individual kinase enzymology and drug discovery in a purified system, with a focus on validated drug targets as benchmarks. We demonstrate that this approach can be adapted to many known kinase substrates and highlight the benefits of using mass spectrometry to measure kinase activity in vitro, including increased sensitivity. We speculate that this approach to measuring kinase activity will be generally applicable across most of the kinome, enabling research on understudied kinases and kinase drug discovery.

Repurposed dihydroorotate dehydrogenase inhibitors with efficacy against drug-resistant Acinetobacter baumannii.

New antimicrobials are needed for the treatment of extensively drug-resistant Acinetobacter baumannii. The de novo pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is a validated drug target for malaria and human autoimmune diseases. We provide genetic evidence that A. baumannii DHODH (AbDHODH) is essential for bacterial survival in rodent infection models. We chemically validate the target by repurposing a unique library of ~450 triazolopyrimidine/imidazopyrimidine analogs developed for our malaria DHODH program to identify 21 compounds with submicromolar activity on AbDHODH. The most potent (DSM186, DHODH IC50 28 nM) had a minimal inhibitory concentration of ≤1 µg/ml against geographically diverse A. baumannii strains, including meropenem-resistant isolates. A structurally related analog (DSM161) with a long in vivo half-life conferred significant protection in the neutropenic mouse thigh infection model. Encouragingly, the development of resistance to these compounds was not identified in vitro or in vivo. Lastly, the X-ray structure of AbDHODH bound to DSM186 was solved to 1.4 Å resolution. These data support the potential of AbDHODH as a drug target for the development of antimicrobials for the treatment of A. baumannii and potentially other high-risk bacterial infections.

Targeting ESR1 mutation-induced transcriptional addiction in breast cancer with BET inhibition.

Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration-approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant-induced endocrine therapy resistance in breast cancer.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Chemical screening identifies novel small molecule activators of natural killer cell cytotoxicity against cancer cells.

Natural killer (NK) cells are cytotoxic lymphocytes that play a major role in the innate immune system. NK cells exhibit potent cytotoxic activity against cancer cells and virally infected cells without antigen priming. These unique cytotoxic properties make NK cells a promising therapeutic against cancer. Limitations of NK cell therapy include deficiencies in high clinical efficacy often due to a need for a high NK cell to target cell ratio to achieve effective killing. In order to address the suboptimal efficacy of current adoptive NK cell therapy, a high throughput screen (HTS) was designed and performed to identify drug-like compounds that increase NK cytotoxic activity against tumor cells without affecting the normal cells. This screen was performed in a 384-well plate format utilizing an expanded primary NK cell product and ovarian cancer cells as a target cell (TC) line. Of the 8000 diverse small molecules screened, 16 hits were identified (0.2% hit rate) based on both a robust Z (RZ) score < -3 and a greater than 10% increase in NK cell killing. A validation screen had a confirmation rate of 70%. Select compounds were further validated and characterized by additional cytotoxicity assays including activity against multiple blood cancer and solid tumor cell lines, with no effect on primary human T cells. This work demonstrates that high-throughput screening can be reliably used to identify compounds that increase NK tumoricidal activity in vitro that can be further investigated and translated for potential clinical application. Précis: Our work led to the identification of promising compound that potently increases NK cell-mediated killing of a variety of different cancer cells, but no impact on the killing of normal cells. This compound demonstrates the utility of this assay.

Structural Insights into Novel 15-Prostaglandin Dehydrogenase Inhibitors.

We discovered SW033291 in a high throughput chemical screen aimed at identifying 15-prostaglandin dehydrogenase (15-PGDH) modulators. The compound exhibited inhibitory activity in in vitro biochemical and cell-based assays of 15-PGDH activity. We subsequently demonstrated that this compound, and several analogs thereof, are effective in in vivo mouse models of bone marrow transplant, colitis, and liver regeneration, where increased levels of PGE2 positively potentiate tissue regeneration. To better understand the binding of SW033291, we carried out docking studies for both the substrate, PGE2, and an inhibitor, SW033291, to 15-PGDH. Our models suggest similarities in the ways that PGE2 and SW033291 interact with key residues in the 15-PGDH-NAD+ complex. We carried out molecular dynamics simulations (MD) of SW033291 bound to this complex, in order to understand the dynamics of the binding interactions for this compound. The butyl side chain (including the sulfoxide) of SW033291 participates in crucial binding interactions that are similar to those observed for the C15-OH and the C16-C20 alkyl chain of PGE2. In addition, interactions with residues Ser138, Tyr151, and Gln148 play key roles in orienting and stabilizing SW033291 in the binding site and lead to enantioselectivity for the R-enantiomer. Finally, we compare the binding mode of (R)-S(O)-SW033291 with the binding interactions of published 15-PGDH inhibitors.

Total synthesis of (+)-spiroindimicin A and congeners unveils their antiparasitic activity.

The spiroindimicins are a unique class of chlorinated indole alkaloids characterized by three heteroaromatic rings structured around a congested spirocyclic stereocenter. Here, we report the first total synthesis of (+)-spiroindimicin A, which bears a challenging C-3'/C-5''-linked spiroindolenine. We detail our initial efforts to effect a biomimetic oxidative spirocyclization from its proposed natural precursor, lynamicin D, and describe how these studies shaped our final abiotic 9-step solution to this complex alkaloid built around a key Pd-catalyzed asymmetric spirocyclization. Scalable access to spiroindimicins A, H, and their congeners has enabled discovery of their activity against several parasites relevant to human health, providing potential starting points for new therapeutics for the neglected tropical diseases leishmaniasis and African sleeping sickness.

Aryl Sulfonamide Inhibits Entry and Replication of Diverse Influenza Viruses via the Hemagglutinin Protein.

Influenza viruses cause approximately half a million deaths every year worldwide. Vaccines are available but partially effective, and the number of antiviral medications is limited. Thus, it is crucial to develop therapeutic strategies to counteract this major pathogen. Influenza viruses enter the host cell via their hemagglutinin (HA) proteins. The HA subtypes of influenza A virus are phylogenetically classified into groups 1 and 2. Here, we identified an inhibitor of the HA protein, a tertiary aryl sulfonamide, that prevents influenza virus entry and replication. This compound shows potent antiviral activity against diverse H1N1, H5N1, and H3N2 influenza viruses encoding HA proteins from both groups 1 and 2. Synthesis of derivatives of this aryl sulfonamide identified moieties important for antiviral activity. This compound may be considered as a lead for drug development with the intent to be used alone or in combination with other influenza A virus antivirals to enhance pan-subtype efficacy.

Novel Antimalarial Tetrazoles and Amides Active against the Hemoglobin Degradation Pathway in Plasmodium falciparum.

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

A Multipronged Approach Establishes Covalent Modification of ß-Tubulin as the Mode of Action of Benzamide Anti-cancer Toxins.

A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A "clickable" benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as ß-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that ß-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with ß-tubulin harboring Cys239, but not ß3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in ß3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in ß-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.