Cell Chemical Biology

Release (egress) of malaria parasites from host red blood cells (RBC) is a protease-dependent process involving breakdown of the RBC cytoskeleton by a parasite cysteine protease-like protein called SERA6. In the penultimate step of the egress cascade, SERA6 undergoes autoproteolytic maturation triggered upon cleavage by a serine protease called SUB1 and requiring interactions between SERA6 and fragments of another parasite protein called MSA180. Egress can be blocked by treatment of intraerythrocytic parasites with small molecules that prevent the autocatalytic SERA6 maturation step, suggesting that SERA6 is a druggable target. Here we describe the development of a cell-free in vitro system that recapitulates SERA6 maturation. We use the assay to confirm the strict requirement for MSA180 in SERA6 maturation by SUB1 and to show that these 3 components are sufficient for SERA6 maturation. Using a synthetic peptide substrate based on a predicted autocatalytic cleavage site we demonstrate that the fully mature SERA6 is an active proteolytic enzyme and we validate improved small molecule inhibitors of SERA6. Our lead inhibitory compound efficiently blocks egress of asexual blood stage parasites, confirming SERA6 as a new potential antimalarial drug target.

Reversible inactivation of protein tyrosine phosphatases by reactive oxygen species (ROS) is essential to the phosphorylation of growth factor receptors. An important outcome of the inactivation of protein tyrosine phosphatase 1B (PTP1B) by ROS involves the conformational change of its phosphotyrosine binding loop which adopts a solvent exposed position in its oxidized form. We previously demonstrated that 14-3-3{zeta} binds to the phosphotyrosine binding loop of the oxidized form of PTP1B. Using a rational approach, we developed a unique protein-protein interaction (PPI) inhibitor peptide derived from the phosphotyrosine binding loop of PTP1B designed to disrupt the interaction between PTP1B and the 14-3-3{zeta}-complex. Exploiting this cell-permeable peptide, we showed decreased association between PTP1B and the 14-3-3{zeta}-complex in cells treated with epidermal growth factor (EGF). We also demonstrated that preventing the association of this 14-3-3{zeta}-complex to PTP1B deterred oxidation and inactivation of PTP1B following EGF receptor (EGFR) activation and generation of ROS. Treating cells with our PPI inhibitor decreased EGFR phosphorylation on PTP1B-specific sites. Furthermore, treating EGFR-driven epidermal cancer cells with our PPI inhibitor also significantly inhibited colony formation and cell viability, consitent with increased activation of PTP1B. These data highlight the ability of PTP1B to downregulate critical signaling pathways in cancer when activated using peptide drugs such as our protein-protein interaction inhibitor. We anticipate that preventing or destabilizing the reversible oxidation of other members of the protein tyrosine phosphatase superfamily using PPI inhibitors may offer a foundation for a broad therapeutic approach to rectify dysregulated signaling pathways in vivo. Significance StatementLimited understanding of redox mechanisms regulating PTP catalytic activity is a major knowledge gap that has hampered our efforts to develop activation strategies. In its reversibly oxidized and inactivated form, conformational changes of PTP1B influence its association with regulatory proteins. We demonstrate that designing a cell-permeable peptide based on a loop of PTP1B that becomes exposed during oxidation can block its interaction with the 14-3-3{zeta}-multiprotein complex and activate the phosphatase. Moreover, activating PTP1B using our protein-protein interaction inhibitor peptide decreases the phosphorylation of its substrate EGFR and decreases the effectiveness of cancer cells to form colonies. This study provides important insights into the therapeutic potential of protein-protein interaction inhibitors that regulate the redox cycle of PTPs to reestablish physiological signaling.

Ferroptosis is a cell death mechanism characterized by the accumulation of iron-catalyzed lipid peroxides in membrane lipid acyl chains and subsequent loss of membrane integrity.1 Despite thorough investigation of its mechanisms in cultured cells, induction of ferroptosis has unresolved clinical utility in cancer therapy. Here, we systematically evaluate ferroptosis induction via multiple mechanisms, in both cell and tumor models, using focused genetic screens, genetic loss-of-function systems, and pharmacological perturbations. Through this analysis we identify cancer cell line subsets with distinct responses to canonical ferroptosis inducers and suppressors and define the underpinnings of each. Inhibition of central in vitro ferroptosis suppressors GPX4, GCLC, or SLC7A11 across these multiple models fails to impact established tumor growth. In contrast, deficiency in the cytosolic thioredoxin reductase and pharmacologic GCLC inhibition potently induces tumor regression and triggers a form of non-ferroptotic cell death regulated by cystine availability and translation. These analyses further reveal that the principal essential function of environmental cystine in cultured cells is to support selenoprotein function, identified through investigating our finding that {beta}-mercaptoethanol supports exponential growth in cystine-free conditions. Thus, while ferroptosis activation may be efficacious alone or in combination with other therapies in specific tumor contexts, cell culture systems greatly overestimate the potential anti-cancer effects of ferroptosis induction via the GPX4 axis.

In the effort to develop efficacious antibacterials that engage targets for which there is no pre-existing resistance, inhibition of the enoyl-acyl carrier protein reductase FabI has shown promise, with triclosan and fabimycin as representative members of the two major drug classes that show activity against important bacterial pathogens. Here, we use a morbidostat and whole genome sequencing to comprehensively evaluate the resistance profiles that arise in pathogenic bacteria in response to these FabI inhibitors. When assessed against E. coli, fabimycin and triclosan were found to induce primarily non-overlapping resistance profiles leading to minimal cross-resistance between the two compounds. Furthermore, in vivo evaluation of the prominent resistant mutants indicates poor fitness, with the most fit mutant still susceptible to fabimycin. Collectively, these results suggest the combination use of two antibiotics that engage different positions on the same target as a means to kill pathogenic bacteria and limit resistance.

Plasmodium falciparum resistance to current first line treatments is threatening at-risk populations and underscores the urgent need for novel therapeutic targets and drugs. P. falciparum pyruvate kinases I and II are two essential enzymes with distinct roles and subcellular localizations within the parasite. PfPyrKI is cytosolic, while PfPyrKII is found in the apicoplast, a specific organelle of Apicomplexa, where it is required for the production of (d)NTPs essential for apicoplast maintenance. We identify skeletocutin E, a Basidiomycete-derived metabolite, as a specific inhibitor of PfPyrKII. Skeletocutin E inhibits in vitro the activity of PfPyrKII with an IC50 of 0.52 {+/-} 0.08 {micro}M through a mixed inhibition mechanism and does not affect the activities of three human pyruvate kinases. Structure-activity relationship analyses using synthetic skeletocutin E analogues allowed us to identify the molecular determinants of this inhibition. Furthermore, determination of the quaternary structure of PfPyrKII by mass photometry, showed that this enzyme exists as monomers, dimers, and tetramers in equal proportions, revealing its singularity compared to other pyruvate kinases. Interestingly, skeletocutin E does not alter the distribution of the complexes, indicating that it does not interact at the subunit interfaces. Importantly, skeletocutin E inhibits P. falciparum growth in both blood and liver stages, with IC values of 3.56 {+/-} 0.50 {micro}M in red blood cells and 3.70 {+/-} 0.74 {micro}M in primary human hepatocytes. Together, these findings establish PfPyrKII as a druggable antimalarial target and identify skeletocutin E as a promising lead compound for the rational development of dual-stage antimalarial therapies.

Neurosteroids are endogenous neuromodulators and emerging therapeutics, but understanding but understanding how these compounds modulate receptor signaling within defined neuronal populations and networks has been limited by an inability to deliver these molecules with receptor-level and cell-type specificity. Here, we developed a neurosteroid DART (Drug Acutely Restricted by Tethering) that combines the GABAA receptor subunit-selectivity of a neuroactive steroid (NAS) with the cell-type specificity of the DART platform. Screening of seventeen NAS analogs identified seven scaffolds suitable for further engineering, and structure-activity analysis revealed that DART linker attachment at the C11 position preserved NAS activity on GABAA receptors, whereas C2 and C17 attachment failed to exhibit activity. Functional profiling of C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The most selective compound, YX85.1DART.2, potentiated GABA-evoked currents in neurons expressing pharmacogenetically isolated 4/{delta}-containing GABAA receptors but not in {gamma}2-expressing neurons. A previously validated BZP.1DART.2 produced complementary selectivity on the two receptor populations. Together, these findings establish new tools for interrogating subunit-specific NAS actions on inhibitory signaling in defined neuronal populations.

The CBL E3 ubiquitin ligase is a critical regulator of tyrosine kinase (TK) signalling. CBL activity is regulated by a feedback loop in which an active TK phosphorylates CBL, relieving its autoinhibited conformation, and allowing ubiquitination of substrates, including TKs, leading to their degradation. Binding of the Src Like Adapter Protein 2 (SLAP2) to CBL can also activate autoinhibited CBL and promote substrate ubiquitination. Using an engineered CBL mutant in the SLAP2 binding interface, termed RE CBL, that mimics activation by SLAP2 binding, we characterized the cellular functions of this CBL activation mechanism. Comparison of wildtype and RE CBL interactomes using MiniTurboID showed extensive and overlapping interaction networks, with a discrete subset of proteins, including the known substrate, epidermal growth factor receptor (EGFR), as well as endocytic factors such as EPS15, in higher abundance with RE CBL compared to wildtype. Consistent with these observations, RE CBL interacted more readily with EGFR, enhanced EGFR internalization, and attenuated downstream signalling compared to WT. In Cbl null hematopoietic cells, RE CBL expression reduced sensitivity to cytokines IL-3 and GM-CSF, and decreased activation of the Src-family kinase Lyn. Furthermore, we optimized and conducted a small molecule screen to identify a group of structurally related compounds that, like SLAP2 binding, promoted CBL activation in vitro. Together these findings provide proof of concept for targeting CBL activity to downregulate TK signalling.

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that bring a ubiquitin E3 ligase into proximity of a target protein to polyubiquitinate and degrade the target. PROTACs act catalytically, offering distinct advantages over conventional inhibitors and are the subject of intense study. The development of PROTACs involves extensive optimization of the chemical moiety linking two different protein-binding chemotypes, often requiring the synthesis, purification and testing of hundreds of PROTAC candidates. We used this approach to rapidly explore the landscape of targeted degradation of four different targets in parallel, combining and comparing a recently reported FBXO22-recruiting chemical warhead with warheads for the commonly used CRBN and VHL E3 ligases. Using a limited number of compounds (175 compounds in total) we observed no FBXO22-dependent degradation of these four targets. However, our libraries generated potent FBXO22 homo-PROTACs inducing self-degradation, as well as CRBN- and VHL-mediated degraders of FBXO22.

Many genetically validated targets in cancer, including the transcription factor {beta}-catenin ({beta}-cat), have historically been viewed as undruggable. Cell-based phenotypic screening of chemical compounds can reveal new biological and pharmacological principles. Natural products are powerful probes because of their superior structural diversity, drug-like properties, and biological activities as compared to unoptimized synthetic compounds. We screened 326,304 natural product mixtures (40,744 extracts and 285,560 fractions derived from them) using mammalian cells expressing an oncogenic version of {beta}-cat fused to a suicide protein. Multiple fractions degraded the {beta}-cat fusion protein or drove it into a compartment where both fusion partners were apparently inactive. The active natural product from one of the latter specifically activates novel, but not classical, protein kinase Cs (PKCs) and thereby relocates {beta}-cat to juxtamembrane vacuolar structures. These findings suggest a path for inactivating oncogenic {beta}-cat and underscore the power of screening natural product collections with robust phenotypic assays.

Phosphatidic acid (PA) is an essential intermediate generated during phospholipase C (PLC) signaling, but its regulation is complex. PA can be generated by ten different diacylglycerol kinase paralogs (DGKs) and two different phospholipase D paralogs (PLDs) in mammals. Because these enzymes are activated under diverse conditions and at various membranes, understanding paralog-specific contributions to PA production is critical for therapeutic development of drugs that modulate the PLC pathway. To address this, we aimed to characterize the paralog specificity of the DGK inhibitors R59022 and BMS-502 against individual DGK paralogs in cellulo. We found that R59022 and BMS-502 both recruited endogenous DGK to the plasma membrane, and inhibited the catalytic fragment of DGK when ectopically localized to the mitochondrial outer membrane. However, at its effective dose, R59022 paradoxically increased PA levels and was cytotoxic, while BMS-502 functioned as a potent and nontoxic inhibitor. Live-cell imaging experiments using BMS-502 with carbachol stimulation of endogenous muscarinic receptors showed that inhibition of both DGK and the PLDs is needed to substantially reduce PA levels during PLC activation. Our findings both identify paralog-specific druggable targets for modulating PLC signaling events, and establish a new platform that translates typical biochemical dose response assays in cellulo.

MicroRNAs (miRNAs) typically regulate gene expression by promoting mRNA degradation, but select miRNAs, such as miR-466l-3p (miR-466), can instead stabilize transcripts in coordination with RNA-binding proteins (RBPs) like HuR. We identify conserved AU-rich elements (cAREs) within the 3'UTRs of IL-17A, GM-CSF, and IL-23A as critical cis-regulatory binding sites where miR-466 facilitates HuR recruitment to promote mRNA stability. Using site-directed mutagenesis, RNA pulldown, and MS2-TRAP assays to capture miRNA-mRNA complexes, we demonstrate that HuR binding depends on prior engagement by miR-466. Disrupting this interaction with rationally designed Target Site Blockers (TSBs) oligonucleotides destabilizes target mRNAs and suppresses cytokine expression in vitro and in vivo. TSBs directed against IL-17A, GM-CSF, and IL-23A selectively blocked miR-466 binding, reduced transcript stability, and lowered cytokine production without affecting unrelated mRNAs. In murine models of LPS-induced inflammation, psoriasis, and autoimmunity, TSBs exhibited therapeutic efficacy and cytokine specificity, outperforming monoclonal antibodies in some settings. Phosphorothioate-modified TSBs enabled systemic delivery and retained activity in human T cells, underscoring translational potential. Similar to antisense oligonucleotides, TSBs trigger RNase H1-mediated degradation while also blocking miRNA-mRNA interactions. These findings establish miR-466-HuR cooperation as a therapeutically targetable axis through TSBs without affecting global miRNA function. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/709388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@82c326org.highwire.dtl.DTLVardef@da15e7org.highwire.dtl.DTLVardef@1d3fc69org.highwire.dtl.DTLVardef@607656_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TEXTBOXMechanism of TSB-mediated disruption of cooperative miRNA-HuR-dependent mRNA stabilizationA: In the canonical model, destabilizing miRNAs (e.g., miR-16) bind to their target sites within the 3'UTR, recruiting the RNA-induced silencing complex (miRISC) to promote mRNA decay or translational repression. B: In contrast, a newly identified class of miRNAs--stabilizing miRNAs (E-miRNAs), such as miR-466l-3p--bind to specific target sequences within AU-rich elements (AREs) in the 3'UTR. This binding facilitates cooperative recruitment of the RNA-binding protein HuR (ELAVL1), resulting in enhanced mRNA stability and/or translation. C: Target site blockers (TSBs) designed to occlude miRNA-binding sites competitively inhibit miRISC loading, thereby disrupting HuR engagement and reversing stabilization. This selective disruption leads to transcript-specific mRNA destabilization without affecting global miRNA function. C_TEXTBOX

N6-methyladenosine (m6A) is a major epitranscriptomic modification that regulates RNA metabolism and affects the replication and latency reversal of human immunodeficiency virus type 1 (HIV-1) in cells. Methyltransferase-like 3 (METTL3) is the principal catalytic enzyme responsible for m6A deposition, and its pharmacological inhibition has emerged as a potential therapeutic strategy for cancer and viral infections. However, the relative potency of METTL3 inhibitors in reducing m6A levels and their effects on HIV-1 latency reversal remain undefined. Here, we compared three commercially available METTL3 inhibitors (STM2457, STM3006, and STC-15) to evaluate their ability to reduce RNA m6A levels, suppress HIV-1 latency reversal, and affect cell viability in latently infected J-Lat cells and primary CD4+ T cells. In J-Lat cells, STM3006 and STC-15 were more potent than STM2457 in reducing RNA m6A levels at 24 and 48 hours post-treatment, as reflected by lower half-maximal inhibitory concentrations (IC50). However, STM3006 and STC-15 exhibited significant cytotoxicity at concentrations above 2 {micro}M at 48 hours post-treatment, whereas STM2457 displayed minimal toxicity across all tested doses. In primary CD4+ T cells from three healthy donors, all three inhibitors reduced RNA m6A levels but induced greater cytotoxicity compared with J-Lat cells, with comparable effects at optimized concentrations. Notably, reduced RNA m6A levels correlated with diminished HIV-1 latency reversal in both J-Lat cells and a primary central memory CD4+ T cell model. Together, these findings demonstrate differential potency and cytotoxicity among METTL3 inhibitors and support a critical role for m6A RNA modification in regulating HIV-1 latency reversal.

PIKfyve is a lipid kinase involved in regulating protein clearance mechanisms and is a promising target for the treatment of neurodegenerative diseases. Here, we present the discovery and optimization of a CNS-penetrant covalent PIKfyve inhibitor, DUN058, which achieves sustained target occupancy in vivo. Covalent screening hits, identified from chemoproteomics experiments performed in live cells, were rapidly optimized to deliver a brain-penetrant covalent inhibitor of PIKfyve. This covalency centered approach employed a suite of mass spectrometry, biochemical and in vivo assays to optimize compound potency, selectivity, and CNS permeability. The target nucleophile, cysteine 1970, is on a flexible loop that appears distal from the kinase active site, highlighting the power of chemoproteomics screening to identify novel nucleophilic amino acids for covalent modification. DUN058 achieves efficient covalency at the target cysteine, as well as highly selective covalent and reversible selectivity profiles. Covalent PIKfyve inhibition results in modulation of downstream pathway activity, including activation of the transcription factor TFEB, upregulation of protein clearance pathways, and increased GPNMB transcription and secretion of exosome markers. When dosed in vivo, DUN058 achieves sustained target occupancy in the brains of mice long after systemic compound clearance, holding promise for achieving a sustained duration of action in the CNS at low doses, without prolonged effects in the periphery. Taken together, the development of DUN058 is a demonstration of chemoproteomics-based discovery for a high value CNS target, providing an orally bioavailable and covalent PIKfyve inhibitor.

Thalidomide is teratogenic in humans but not in rodents due to species-specific differences in the sequence of Cereblon (CRBN), an E3 ubiquitin ligase targeted by thalidomide and its derivative molecular glue degraders (MGDs). This species divergence has hindered the accurate prediction of MGD-induced toxicities in standard laboratory animals. GSPT1 MGDs, such as CC-90009, have shown potent anticancer activities in preclinical models and leukemia patients; however, their clinical development was challenging due to severe adverse effects. This highlights the critical need to characterize on-target toxicities in relevant animal models to exploit the therapeutic safety of this class of MGDs. Here, we generated humanized CrbnV380E and CrbnV380E/I391V knock-in mouse strains, in combination with a degradation-resistant Gspt1G574N strain, to interrogate the in vivo on-target effects of CC-90009 and its analog CC-885. We found that targeted GSPT1 depletion in mice led to rapid mortality, preceded by multiple dysfunctions including intestinal obstruction, liver damage, splenic atrophy, and hematological abnormalities. Remarkably, these toxicities, along with the underlying transcriptional perturbations, were completely rescued by the undegradable Gspt1G574N mutant, establishing a definitive causal link between GSPT1 degradation and systemic injury. Induced proximity and degradation proteomic analyses revealed that GSPT1 loss triggered a secondary downregulation of many proteins, including MYC, PLK1 and CDK4, which were not directly recruited by these MGDs to CRBN. Collectively, our data define the in vivo on-target toxicities associated with endogenous GSPT1 degradation and provide a genetic framework to guide the preclinical safety evaluation of CRBN-based MGD therapeutics.

The targeted modulation of gene expression with bifunctional small molecules enables the precise control of cellular and biological processes. To screen for ligands that could be used to induce gene expression, we conjugated the high affinity FKBP(F36V) binder, AP1867, to known high-affinity binders of activating epigenetic machinery. We tested these bifunctionals in a FKBP(F36V)-tagged transcription factor reporter system and found bifunctional induced transactivation is relatively common, being observed for bifunctionals with BET ligand JQ1, p300/CBP ligand GNE-781, CDK9 ligand SNS-032, and BRD9 ligand iBRD9. aTAG-2 (mAP1867-C8-GNE781) was identified as the strongest and most potent transactivator, possessing single-digit nanomolar activity. When tested in models where oncogenic RNA binding protein-transcription factor fusion proteins have been FKBP(F36V)-tagged, we unexpectedly observed rapid collapse of the fusion transcriptional program. In a tagged Ewing sarcoma model, aTAG-2 exhibits at least three distinct mechanisms of action: i) RIPTAC mediated p300/CBP inhibition, ii) ubiquitination- and ternary complex-dependent EWS/FLI degradation, and iii) replacement of p300 with CBP at EWS/FLI bound chromatin loci. Together, these data establish bifunctionals targeting p300/CBP that toggle between a program of ultra-potent transactivation and repression depending on cellular context. Overall demonstrating that induced proximity with a given ligand does not encode a fixed functional outcome.

Western-style diets promote chronic metabolic inflammation and dyslipidemia, yet safe interventions that restore immunometabolic homeostasis remain limited. Pyrroloquinoline quinone (PQQ) is a naturally occurring redox cofactor with antioxidant and metabolic regulatory properties, but its systemic effects in translational preclinical models are poorly defined. Here, we examined the impact of short-term PQQ supplementation in obese adult female olive baboons (Papio anubis) chronically fed a Western diet. Using a human-equivalent dose administered for 30 days, we found that PQQ supplementation significantly reduced circulating markers of systemic inflammation and cholesterol in Western-diet-fed animals, lowering circulating C-reactive protein, soluble CD163, and atherogenic lipoprotein fractions independent of changes in adiposity. Proteomic and pathway analyses of circulating proteins in plasma and serum revealed suppression of complement, thrombo-inflammatory, and extracellular matrix remodeling pathways, alongside enhanced lipoprotein assembly, remodeling, and clearance. Network analyses identified restoration of neurotrophic tyrosine kinase receptor 1 (NTRK1)- and forkhead box A2 (FOXA2)-regulated signaling as central features of the PQQ response, accompanied by inhibition of pro-fibrotic, xenobiotic, and inflammatory pathways, as well as predicted activation of liver X receptor (LXR)- and insulin growth factor (IGF)-associated metabolic programs. These findings demonstrate that PQQ rapidly reprograms systemic immunometabolic networks in a nonhuman primate model of diet-induced metabolic stress, highlighting FOXA2- and neurotrophin-associated pathways as novel targets of PQQs action.

PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) is an antimicrobial discovery platform based on chemical-genetic interaction (CGI) profiling of compounds against a pool of Mycobacterium tuberculosis (Mtb) hypomorphs, each depleted of an essential gene. From prior screening data, we have now identified a novel N-oxolan-3-yl pyrazole carboxamide inhibitor (BRD1554) that had increased, selective activity against strains depleted of polyketide synthase 13 (Pks13), an essential enzyme in mycolic acid synthesis, and Rv2581c, an uncharacterized protein similar to glyoxylase II enzymes. Perturbagen CLass (PCL) analysis, a reference-based approach to mechanism of action (MOA) assignment from PROSPECT, predicted Pks13, a polyketide synthase with five catalytic domains responsible for the terminal condensation step in mycolic acid biosynthesis, was the likely target, potentially implicating the thioesterase domain. We synthesized a more active analogue while assigning the absolute stereochemistry of the active diastereomer, resulting in 1554-06-3R,4S with an MIC90 of 3.0 {micro}M against Mtb H37Rv. Exposure to 1554-06 led to the upregulation of the pks13 operon along with the iniBAC operon and other genes linked to mycolic acid synthesis. Isolation of mutants resistant to 1554-06 revealed single nucleotide polymorphisms in the thioesterase domain of Pks13. Finally, we biochemically confirmed that 1554-06 inhibits the activity of recombinant Pks13 thioesterase domain, with computational docking of 1554-06 steroisomers consistent with the stereospecific activity seen in whole cell assays. We found unique chemical genetic interactions between inhibitors of the different Pks13 domains and different detoxifying enzymes of Mtb, thus revealing novel gene-gene interactions. These results highlight how PROSPECT can not only immediately reveal, with domain-level resolution, the MOA of new whole-cell active chemical inhibitors of Mtb, allowing the integration of biological insight into compound triage and accelerated early development, but can also illuminate genetic interactions linked to those mechanisms that could inform predictions of synergy for antitubercular drug development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/704361v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@9a4f38org.highwire.dtl.DTLVardef@c70cd2org.highwire.dtl.DTLVardef@1ac2e1org.highwire.dtl.DTLVardef@f06df0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Base excision repair (BER) is the primary pathway that removes oxidatively-induced DNA base damage from the nuclear and mitochondrial genomes, with 8-oxoguanine DNA glycosylase (OGG1) initiating repair at the two most frequently-formed base lesions: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoGua) and 2,6-diamino-4-oxo-5-formamidopyrimidine (FapyGua). Humans expressing a catalytically-compromised variant of OGG1 (S326C) are at increased risk for type 2 diabetes, Alzheimers disease, and Parkinsons disease. To potentially enhance the overall catalytic efficiency of this variant, a prior medicinal chemistry screen discovered seven chemically distinct agonists of OGG1 that stimulated activity in vitro and attenuated a paraquat (PQ) challenge in cultured cells. Herein, we developed structure-activity relationships around one specific core structure, F01. Using fluorescence-based DNA cleavage assays, we assessed the abilities of these compounds to stimulate the overall rate of OGG1 catalysis. Multiple compounds were identified that increased OGG1 activity on DNAs containing a site-specific 8-oxoGua by 2-fold or greater, with 9 compounds showing EC50 concentrations lower than F01 and were specific for OGG1. Selected agonists were shown to enhance OGG1-catalyzed release of 8-oxoGua and FapyGua from {gamma}-irradiated high-molecular-weight DNA using gas chromatography tandem mass spectrometry analyses. Since these assays did not reveal which step in the overall reaction was stimulated, we used a separation-of-function OGG1 mutant that possessed glycosylase, but not abasic-site (AP) lyase activity to demonstrate that the glycosylase step was not enhanced. In contrast, all agonists stimulated the AP lyase activity to levels equal to or greater than the magnitude of stimulation observed for overall chemistry, implicating agonist-mediated turnover as a potential contributor to the overall rate stimulation. The biological activities of selected agonists were evaluated in OGG1-deficient Kasumi-1 cells under conditions of paraquat (PQ)-induced oxidative stress, with several compounds mitigating PQ challenge.

Toxoplasma gondii is a globally important intracellular parasite, and treatment regimens are limited by the failure of drugs to target latent tissue cysts. Developing new candidates for treatment also needs to address the potential for resistance to arise. Here, we developed a Minimum Inoculum for Resistance (MIR) assay as a quantitative metric for evaluating inhibitors of T. gondii. The MIR assay, adapted from assays used in malaria drug discovery, measures the frequency for pre-existing resistance alleles by exposing different sized parasite populations to drug pressure. We profiled a series of bicyclic pyrrolidone analogs that inhibit phenylalanine tRNA synthetase (PheRS). We demonstrate that these inhibitors require higher inocula to lead to parasite resistance (up to > 108 parasites) in comparison with an inhibitor of DNA synthesis, and that MIR values vary across inhibitors with closely related chemical structures. Clonal analysis of resistant parasites emerging from MIR assays revealed both new and previously identified resistance conferring mutations in TgPheRS, and structural modeling revealed their potential impact the enzyme active site. The MIR assay provides a functional benchmark to compare new and existing inhibitors, allowing for rational prioritization of lead compounds with a high genetic barrier to resistance.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein cytokine with therapeutic potential in cancer and neutropenia treatment. While glycosylation of GM-CSF reduces immunogenicity and enhances serum bioavailability, it can also diminish receptor binding and bioactivity. Based on transcriptomic analysis of human T lymphocytes reported previously, GM-CSF-producing cells exhibit elevated expression of Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase (MGAT5), which encodes N-acetylglucosaminyltransferase V, an enzyme involved in N-glycan branching. Given this role of MGAT5 in glycosylation, we produced GM-CSF variants using glycoengineered Chinese hamster ovary cells to generate diverse glycoforms and assessed their bioactivity. Testing their activity on TF-1 cell proliferation, we found that decreases in GM-CSF N-glycan branching significantly suppressed its activity. These findings underscore the importance of glycosylation in modulating the efficacy and safety of GM-CSF-based therapeutics, suggesting that precise glycoengineering may be key to optimizing GM-CSF performance in clinical applications.