In Silico Drug Screening Analysis against the Overexpression of PGAM1 Gene in Different Cancer Treatments.

Phosphoglycerate mutase 1 (PGAM1) is considered as a novel target for multiple types of cancer drugs for the upregulation in tumor, cell prefoliation, and cell migration. During aerobic glycolysis, PGAM1 plays a critical role in cancer cell metabolism by catalyzing the conversion of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG). In this computational-based study, the molecular docking approach was used with the best binding active sites of PGAM1 to screen 5,000 Chinese medicinal phytochemical library. The docking results were three ligands with docking score, RMSD-refine, and residues. Docking scores were -16.57, -15.22, and -15.74. RMSD values were 0.87, 2.40, and 0.98, and binding site residues were Arg 191, Arg 191, Arg 116, Arg 90, Arg 10, and Tyr 92. The best compounds were subjected to ADMETsar, ProTox-2 server, and Molinspiration analysis to evaluate the toxicological and drug likeliness potential of such selected compounds. The UCSF-Chimera tool was used to visualize the results, which shows that the three medicinal compounds named N-Nitrosohexamethyleneimine, Subtrifloralactone-K, and Kanzonol-N in chain-A were successfully binding with the active pockets of PGAM1. The study might facilitate identifying the hit molecules that could be beneficial in the development of antidrugs against various types of cancer treatment. These hit phytochemicals could be beneficial for further investigation of a novel target for cancer.

In silico-based identification of phytochemicals as novel human phosphoglycerate mutase 1 (PGAM1) inhibitors for cancer therapy.

Targeting cancer-specific metabolic and mitochondrial remodeling has emerged as a novel and selective strategy for cancer therapy during recent years. Phosphoglycerate Mutase 1 (PGAM1) is an important glycolytic enzyme that catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate and plays a critical role in cancer progression by coordinating glycolysis and biosynthesis. PGAM1 has been reported to be over expressed in a variety of cancer types and its inhibition results in decreased tumor growth and metastasis. Recently, there has been a growing interest in identification and characterization of novel PGAM1 inhibitors for the treatment of cancer. In the current study, in silico tools were used to find out natural inhibitors of PGAM1. For docking studies, a database of 5006 phytochemicals were docked against PGAM1, using MOE software in order to identify the compounds which show better binding affinities than PGMI-004A. Out of 5006 compounds screened, eight compounds (1,3-cyclopentanedione, glyflavanone B, 6-demethoxytangeretin, gnaphaliin, lantalucratin A and -(-) morelensin, abyssinin II and monotesone-A) showed significant binding affinity with PGAMI active site. Further, the eight selected compounds were evaluated for different pharmacokinetics parameters using admetSAR, the obtained results demonstrated that none of these hit compounds violated Lipinski's drug rule of 5 and all the hit compounds possess favorable ADMET properties. This study has unveiled the potential of phytochemicals that could serve as probable lead candidates for the development of PGAM1 inhibitors as anti-cancer agents.

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba.

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Design, synthesis, and biological evaluation of 1,3,6,7-tetrahydroxyxanthone derivatives as phosphoglycerate mutase 1 inhibitors.

Phosphoglycerate mutase 1 (PGAM1) is a promising target for cancer treatment. Herein, we found that α-mangostin and γ-mangostin exhibited moderate PGAM1 inhibitory activities, with IC50 of 7.2 µM and 1.2 µM, respectively. Based on α-mangostin, a series of 1,3,6,7-tetrahydroxyxanthone derivatives were designed, synthesized and evaluated in vitro for PGAM1 inhibition. The significant structure-activity relationships (SAR) and a fresh binding mode of this kind of new compounds were also clearly described. This study provides valuable information for further optimization of PGAM1 inhibitors with 1,3,6,7-tetrahydroxyxanthone backbone or de novo design of novel inhibitor.

HKB99, an allosteric inhibitor of phosphoglycerate mutase 1, suppresses invasive pseudopodia formation and upregulates plasminogen activator inhibitor-2 in erlotinib-resistant non-small cell lung cancer cells.

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, remains a major challenge in the targeted therapy of non-small cell lung cancer (NSCLC). HKB99 is a novel allosteric inhibitor of phosphoglycerate mutase 1 (PGAM1) that preferentially suppresses cell proliferation and induces more apoptosis in acquired erlotinib-resistant HCC827ER cells compared with its parental HCC827 cells. In this study we identified the molecular biomarkers for HKB99 response in erlotinib-resistant HCC827ER cells. We showed that HCC827ER cells displayed enhanced invasive pseudopodia structures as well as downregulated plasminogen activator inhibitor-2 (PAI-2). Meanwhile, PAI-2 knockdown by siPAI-2 candidates decreased the sensitivity of HCC827 parental cells to erlotinib. Moreover, HKB99 (5 µM) preferentially inhibited the invasive pseudopodia formation and increased the level of PAI-2 in HCC827ER cells. Collectively, this study provides new insight into the role of PAI-2 in regulating the sensitivity of erlotinib resistant NSCLC cells to PGAM1 inhibitor. Furthermore, PAI-2 level might be considered as a potential biomarker for predicting the efficacy of the PGAM1 allosteric inhibitor on the erlotinib resistant NSCLC cells.

A Macrocyclic Peptide Library with a Structurally Constrained Cyclopropane-containing Building Block Leads to Thiol-independent Inhibitors of Phosphoglycerate Mutase.

Here we report the construction of an mRNA-encoded library of thioether-closed macrocyclic peptides by using an N-chloroacetyl-cyclopropane-containing exotic initiator whose structure is more constrained than the ordinary N-chloroacetyl-α-amino acid initiators. The use of such an initiator has led to a macrocycle library with significantly suppressed population of lariat-shaped species compared with the conventional libraries. We previously used a conventional library and identified a small lariat thioether-macrocycle with a tail peptide with a C-terminal free Cys whose sidechain plays an essential role in potent inhibitory activity against a parasitic model enzyme, phosphoglycerate mutase. On the other hand, the cyclopropane-containing macrocycle library has yielded a larger thioether-macrocycle lacking a free Cys residue, which exhibits potent inhibitory activity to the same enzyme with a different mode of action. This result indicates that such a cyclopropane-containing macrocycle library would allow us to access mechanistically distinct macrocycles.

Site-Selective Phosphoglycerate Mutase 1 Acetylation by a Small Molecule.

Post-translational modifications play vital roles in fine-tuning a myriad of physiological processes, and one of the most important modifications is acetylation. Here, we report a ligand-directed site-selective acetylation using KHAc, a derivative of a phosphoglycerate mutase 1 (PGAM1) inhibitor. KHAc binds to PGAM1 and transfers its acetyl group to the ε-NH2 of Lys100 to inactivate the enzyme. The acetyl transfer process was visualized by time-resolved crystallography, demonstrating that the transfer is driven by proximity effects. KHAc was capable of selectively and effectively acetylating Lys100 of PGAM1 in cultured human cells, accompanied by inhibited F-actin formation. Similar strategies could be used for exogenous control of other lysine post-translational modifications.

A Novel Allosteric Inhibitor of Phosphoglycerate Mutase 1 Suppresses Growth and Metastasis of Non-Small-Cell Lung Cancer.

Phosphoglycerate mutase 1 (PGAM1) plays a pivotal role in cancer metabolism and tumor progression via its metabolic activity and interaction with other proteins like α-smooth muscle actin (ACTA2). Allosteric regulation is considered to be an innovative strategy to discover a highly selective and potent inhibitor targeting PGAM1. Here, we identified a novel PGAM1 allosteric inhibitor, HKB99, via structure-based optimization. HKB99 acted to allosterically block conformational change of PGAM1 during catalytic process and PGAM1-ACTA2 interaction. HKB99 suppressed tumor growth and metastasis and overcame erlotinib resistance in non-small-cell lung cancer (NSCLC). Mechanistically, HKB99 enhanced the oxidative stress and altered multiple signaling pathways including the activation of JNK/c-Jun and suppression of AKT and ERK. Collectively, the study highlights the potential of PGAM1 as a therapeutic target in NSCLC and reveals a distinct mechanism by which HKB99 inhibits both metabolic activity and nonmetabolic function of PGAM1 by allosteric regulation.

An allosteric PGAM1 inhibitor effectively suppresses pancreatic ductal adenocarcinoma.

Glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) plays a critical role in cancer metabolism by coordinating glycolysis and biosynthesis. A well-validated PGAM1 inhibitor, however, has not been reported for treating pancreatic ductal adenocarcinoma (PDAC), which is one of the deadliest malignancies worldwide. By uncovering the elevated PGAM1 expressions were statistically related to worse prognosis of PDAC in a cohort of 50 patients, we developed a series of allosteric PGAM1 inhibitors by structure-guided optimization. The compound KH3 significantly suppressed proliferation of various PDAC cells by down-regulating the levels of glycolysis and mitochondrial respiration in correlation with PGAM1 expression. Similar to PGAM1 depletion, KH3 dramatically hampered the canonic pathways highly involved in cancer metabolism and development. Additionally, we observed the shared expression profiles of several signature pathways at 12 h after treatment in multiple PDAC primary cells of which the matched patient-derived xenograft (PDX) models responded similarly to KH3 in the 2 wk treatment. The better responses to KH3 in PDXs were associated with higher expression of PGAM1 and longer/stronger suppressions of cancer metabolic pathways. Taken together, our findings demonstrate a strategy of targeting cancer metabolism by PGAM1 inhibition in PDAC. Also, this work provided "proof of concept" for the potential application of metabolic treatment in clinical practice.

Development of Anthraquinone Analogues as Phosphoglycerate Mutase 1 Inhibitors.

Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis and biosynthesis to promote cancer cell proliferation, and is believed to be a promising target for cancer therapy. Herein, based on the anthraquinone scaffold, we synthesized 31 anthraquinone derivatives and investigated the structure-activity relationship (SAR). The 3-substitient of sulfonamide on the anthraquinone scaffold was essential for maintaining potency and the modifications of the hydroxyl of alizarin would cause a sharp decrease in potency. In the meantime, we determined the co-crystal structure of PGAM1 and one of the anthraquinone inhibitors 9i with IC50 value of 0.27 µM. The co-crystal structure revealed that F22, K100 and R116 of PGAM1 were critical residues for the binding of inhibitors which further validated the SAR. Consistent with the crystal structure, a competitive assay illustrated that compound 9i was a noncompetitive inhibitor. In addition, compound 9i effectively restrained different lung cancer cells proliferation in vitro. Taken together, this work provides reliable guide for future development of PGAM1 inhibitors and compound 9i may act as a new leading compound for further optimization.

Synthesis and biological evaluation of anthraquinone derivatives as allosteric phosphoglycerate mutase 1 inhibitors for cancer treatment.

Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis, pentose phosphate pathway, and serine synthesis to promote tumor growth through the regulation of its substrate 3-phosphoglycerate (3 PG) and product 2-phosphoglycerate (2 PG). Herein, based on our previously reported PGAM1 inhibitor PGMI-004A, we have developed anthraquinone derivatives as novel allosteric PGAM1 inhibitors and the structure-activity relationship (SAR) was investigated. In addition, we determined the co-crystal structure of PGAM1 and the inhibitor 8g, demonstrating that the inhibitor was located at a novel allosteric site. Among the derivatives, compound 8t was selected for further study, with IC50 values of 0.25 and approximately 5 µM in enzymatic and cell-based assays, respectively. Mechanistically, compound 8t reduced the glycolysis and oxygen consumption rate in cancer cells, which led to decreased adenosine 5'-triphosphate (ATP) production and subsequent 5' adenosine monophosphate-activated protein kinase (AMPK) activation. The inhibitor 8t also exhibited good efficacy in delaying tumor growth in H1299 xenograft model without obvious toxicity. Taken together, this proof-of-principle work further validates PGAM1 as a potential target for cancer therapy and provides useful information on anti-tumor drug discovery targeting PGAM1.

Inhibition of Phosphoglycerate Mutase 5 Reduces Necroptosis in Rat Hearts Following Ischemia/Reperfusion Through Suppression of Dynamin-Related Protein 1.

PURPOSE: Necroptosis is an important form of cell death following myocardial ischemia/reperfusion (I/R) and phosphoglycerate mutase 5 (PGAM5) functions as the convergent point for multiple necrosis pathways. This study aims to investigate whether inhibition of PGAM5 could reduce I/R-induced myocardial necroptosis and the underlying mechanisms. METHODS: The SD rat hearts (or H9c2 cells) were subjected to 1-h ischemia (or 10-h hypoxia) plus 3-h reperfusion (or 4-h reoxygenation) to establish the I/R (or H/R) injury model. The myocardial injury was assessed by the methods of biochemistry, H&E (hematoxylin and eosin), and PI/DAPI (propidium iodide/4',6-diamidino-2-phenylindole) staining, respectively. Drug interventions or gene knockdown was used to verify the role of PGAM5 in I/R (or H/R)-induced myocardial necroptosis and possible mechanisms. RESULTS: The I/R-treated heart showed the injuries (increase in infarct size and creatine kinase release), upregulation of PGAM5, dynamin-related protein 1 (Drp1), p-Drp1-S616, and necroptosis-relevant proteins (RIPK1/RIPK3, receptor-interacting protein kinase 1/3; MLKL, mixed lineage kinase domain-like); these phenomena were attenuated by inhibition of PGAM5 or RIPK1. In H9c2 cells, H/R treatment elevated the levels of PGAM5, RIPK1, RIPK3, MLKL, Drp1, and p-Drp1-S616 and induced mitochondrial dysfunctions (elevation in mitochondrial membrane potential and ROS level) and cellular necrosis (increase in LDH release and the ratio of PI+/DAPI+ cells); these effects were blocked by inhibition or knockdown of PGAM5. CONCLUSIONS: Inhibition of PGAM5 can reduce necroptosis in I/R-treated rat hearts through suppression of Drp1; there is a positive feedback between RIPK1 and PGAM5, and PGAM5 might serve as a novel therapeutic target for prevention of myocardial I/R injury.

Virtual Screening, Molecular Dynamics and ADME-Tox Tools for Finding Potential Inhibitors of Phosphoglycerate Mutase 1 from Plasmodium falciparum.

BACKGROUND: Nowadays, malaria is still one of the most important and lethal diseases worldwide, causing 445,000 deaths in a year. Due to the actual treatment resistance, there is an emergency to find new drugs. OBJECTIVE: The aim of this work was to find potential inhibitors of phosphoglycerate mutase 1 from P. falciparum. RESULTS: Through virtual screening of a chemical library of 15,123 small molecules, analyzed by two programs, four potential inhibitors of phosphoglycerate mutase 1 from P. falciparum were found: ZINC64219552, ZINC39095354, ZINC04593310, and ZINC04343691; their binding energies in SP mode were -7.3, -7.41, -7.4, and -7.18 kcal/mol respectively. Molecular dynamic analysis revealed that these molecules interact with residues important for enzyme catalysis and molecule ZINC04343691 provoked the highest structural changes. Physiochemical and toxicological profiles evaluation of these inhibitors with ADME-Tox method suggested that they can be considered as potential drugs. Furthermore, analysis of human PGAM-B suggested that these molecules could be selective for the parasitic enzyme. CONCLUSION: The compounds reported here are the first selective potential inhibitors of phosphoglycerate mutase 1 from P. falciparum, and can serve as a starting point in the search of a new chemotherapy against malaria.

The Design and Synthesis of N-Xanthone Benzenesulfonamides as Novel Phosphoglycerate Mutase 1 (PGAM1) Inhibitors.

Upregulation of phosphoglycerate mutase 1 (PGAM1) has been identified as one common phenomenon in a variety of cancers. Inhibition of PGAM1 provides a new promising therapeutic strategy for cancer treatment. Herein, based on our previous work, a series of new N-xanthone benzenesulfonamides were discovered as novel PGAM1 inhibitors. The representative molecule 15h, with an IC50 of 2.1 µM, showed an enhanced PGAM1 inhibitory activity and higher enzyme inhibitory specificity compared to PGMI-004A, as well as a slightly improved antiproliferative activity.

Xanthone derivatives as phosphoglycerate mutase 1 inhibitors: Design, synthesis, and biological evaluation.

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that dynamically converts 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG), which was upregulated to coordinate glycolysis, pentose phosphate pathway (PPP) and serine biosynthesis to promote cancer cell proliferation and tumor growth in a variety of cancers. However, only a few inhibitors of PGAM1 have been reported with poor molecular or cellular efficacy. In this paper, a series of xanthone derivatives were discovered as novel PGAM1 inhibitors through scaffold hopping and sulfonamide reversal strategy based on the lead compound PGMI-004A. Most xanthone derivatives showed higher potency against PGAM1 than PGMI-004A and exhibited moderate anti-proliferation activity on different cancer cell lines.

Conformation and dynamics of the C-terminal region in human phosphoglycerate mutase 1.

Phosphoglycerate mutase 1 (PGAM1), an important enzyme in glycolysis, is overexpressed in a number of human cancers, thus has been proposed as a promising metabolic target for cancer treatments. The C-terminal portion of the available crystal structures of PGAM1 and its homologous proteins is partially disordered, as evidenced by weak electron density. In this study, we identified the conformational behavior of the C-terminal region of PGAM1 as well as its role during the catalytic cycle. Using the PONDR-FIT server, we demonstrated that the C-terminal region was intrinsically disordered. We applied the Monte Carlo (MC) method to explore the conformational space of the C-terminus and conducted a series of explicit-solvent molecular dynamics (MD) simulations, and revealed that the C-terminal region is inherently dynamic; large-scale conformational changes in the C-terminal segment led to the structural transition of PGAM1 from the closed state to the open state. Furthermore, the C-terminal segment influenced 2,3-bisphosphoglycerate (2,3-BPG) binding. The proposed swing model illustrated a critical role of the C-terminus in the catalytic cycle through the conformational changes. In conclusion, the C-terminal region induces large movements of PGAM1 from the closed state to the open state and influences cofactor binding during the catalytic cycle. This report describes the dynamic features of the C-terminal region in detail and should aid in design of novel and efficient inhibitors of PGAM1. A swing mechanism of the C-terminal region is proposed, to facilitate further studies of the catalytic mechanism and the physiological functions of its homologues.

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases.

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.

Phosphoglycerate mutase 1 regulates dNTP pool and promotes homologous recombination repair in cancer cells.

Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet their molecular mechanisms remain poorly understood. Phosphoglycerate mutase 1 (PGAM1) is an important glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway, and serine biosynthesis in cancer cells. Herein, we report that PGAM1 is required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs) caused by DNA-damaging agents. Mechanistically, PGAM1 facilitates DSB end resection by regulating the stability of CTBP-interacting protein (CtIP). Knockdown of PGAM1 in cancer cells accelerates CtIP degradation through deprivation of the intracellular deoxyribonucleotide triphosphate pool and associated activation of the p53/p73 pathway. Enzymatic inhibition of PGAM1 decreases CtIP protein levels, impairs HR repair, and hence sensitizes BRCA1/2-proficient breast cancer to poly(ADP-ribose) polymerase (PARP) inhibitors. Together, this study identifies a metabolically dependent function of PGAM1 in promoting HR repair and reveals a potential therapeutic opportunity for PGAM1 inhibitors in combination with PARP inhibitors.

Oxidative stress activates SIRT2 to deacetylate and stimulate phosphoglycerate mutase.

Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.

Cofactor-independent phosphoglycerate mutase from nematodes has limited druggability, as revealed by two high-throughput screens.

Cofactor-independent phosphoglycerate mutase (iPGAM) is essential for the growth of C. elegans but is absent from humans, suggesting its potential as a drug target in parasitic nematodes such as Brugia malayi, a cause of lymphatic filariasis (LF). iPGAM's active site is small and hydrophilic, implying that it may not be druggable, but another binding site might permit allosteric inhibition. As a comprehensive assessment of iPGAM's druggability, high-throughput screening (HTS) was conducted at two different locations: ∼220,000 compounds were tested against the C. elegans iPGAM by Genzyme Corporation, and ∼160,000 compounds were screened against the B. malayi iPGAM at the National Center for Drug Screening in Shanghai. iPGAM's catalytic activity was coupled to downstream glycolytic enzymes, resulting in NADH consumption, as monitored by a decline in visible-light absorbance at 340 nm. This assay performed well in both screens (Z'-factor >0.50) and identified two novel inhibitors that may be useful as chemical probes. However, these compounds have very modest potency against the B. malayi iPGAM (IC50 >10 µM) and represent isolated singleton hits rather than members of a common scaffold. Thus, despite the other appealing properties of the nematode iPGAMs, their low druggability makes them challenging to pursue as drug targets. This study illustrates a "druggability paradox" of target-based drug discovery: proteins are generally unsuitable for resource-intensive HTS unless they are considered druggable, yet druggability is often difficult to predict in the absence of HTS data.