Ubiquitin-Specific Protease 18 (USP18) is a deISGylation enzyme and antineoplastic target. To develop USP18 inhibitors, an enzymatically active human recombinant USP18 protein was engineered suitable for high-throughput screening of ~80,000 chemical compounds. Three of them substantially inhibited USP18 enzymatic activity, with β-lapachone having prominent antineoplastic activity. Independent β-lapachone treatments of murine and human lung cancer cell lines statistically significantly reduced proliferation and increased apoptosis. Gain of USP18 expression antagonized these effects. β-Lapachone treatments statistically significantly repressed lung cancer xenograft growth. β-Lapachone increased reactive oxygen species (ROS), but antineoplastic effects occurred at dosages with negligible ROS production. ROS scavenger treatments did not rescue β-lapachone effects at these concentrations, consistent with an ROS-independent mechanism. IFN-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 cotransfection antagonized this activity. β-Lapachone treatments increased global ISGylation. RNA-seq of lung cancer cells engineered with or without enhanced USP18 expression showed specific pathways affected by β-lapachone treatment. Proteomic analysis of these treated cells revealed known and new ISGylated proteins. In silico modeling identified a unique USP18 pocket where these USP18 inhibitors bind. Engineered mutation of this pocket disrupted β-lapachone activity. Taken together, β-lapachone is an antineoplastic tool compound useful for USP18 inhibitor development.

Using an easily scalable SARS-CoV-2 main protease (M-Pro) substrate, high-throughput screening of similar to 75,000 compounds drawn from diversity and fragment libraries was conducted. Based on screening results, 40 inhibitors were confirmed with IC50 values below 30 mu M. Four hits were revalidated, and their M-Pro complexes were characterized by X-ray crystallography. For two inhibitors, CDI5573 and CDI8009, clear active-site electron density revealed covalent modification of Cys145, the enzyme's catalytic cysteine. In both structures, His41 of the catalytic dyad reorients from its native hydrogen-bonding pose with Cys145 to engage in pi-stacking with the inhibitors' aromatic rings. The two covalent adducts arise from distinct chemical mechanisms. CDI5573, which is a 3-nitro-1,2,5-oxazole derivative, reacts through nucleophilic aromatic substitution (SNAr) to replace one of the nitro groups by Cys145, whereas CDI8009 undergoes nucleophilic acyl substitution to form a thioester with Cys145. Structure-guided optimization of CDI5573 yielded new analogs, including one inhibitor with an IC50 of 15 nM, placing this molecule among the most potent inhibitors developed for M-Pro. These findings introduce new covalent chemotypes with diverse modes of engagement for M-Pro, expand the accessible space for this essential viral enzyme, provide leads with the potential to complement existing COVID-19 therapeutics and mitigate viral resistance, and guide the targeting of other cysteine enzymes for drug discovery.

TRIP13, a promising target for cancer therapy, has been identified as a key regulator of the mitotic checkpoint. Overexpression of TRIP13 is associated with poor clinical outcomes in various cancers. Inhibition of TRIP13 has the potential to address therapeutic challenges in cancer, particularly in therapy-resistant and Rb-deficient cancers. Despite the potential therapeutic benefits of TRIP13 inhibition, the development of TRIP13 inhibitors has been hindered by the lack of a robust high-throughput screening (HTS) assay. We developed a luminescence-based biochemical assay for TRIP13 activity to address this challenge using the ADP-Glo detection system. This assay offers high sensitivity, low background signal, and ease of automation, making it ideal for HTS applications. A pilot screen of kinase-focused inhibitors library and a large-scale screen of 4000 additional compounds demonstrated the assay's robust performance with a z'-factor exceeding 0.85 and a signal-to-background (S/B) ratio near 6. From the 50 initial hits, rigorous validation identified anlotinib as the most potent TRIP13 inhibitor with an IC50 of 5 μM. A cellular thermal shift assay (CETSA) confirmed the direct binding of anlotinib to TRIP13, validating the potential of our biochemical assay for identifying novel TRIP13 inhibitors. Our study provides a valuable tool for discovering novel TRIP13 inhibitors and advances our understanding of the therapeutic potential of targeting TRIP13 in cancer.

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the α-kinase family of atypical serine/threonine kinases, phosphorylates eEF-2 to slow ribosomal translocation and modulate translational elongation. eEF-2K activation requires Ca2+/calmodulin (CaM) and integrates upstream signals through specific sites within an intrinsically disordered regulatory loop (R-loop; ∼321-520) that links the α-kinase core to a C-terminal domain. Unlike canonical CaM-dependent kinases that are activated by displacement of an autoinhibitory segment that occludes the active site, eEF-2K is activated by CaM-driven stabilization of an active state; Ca2+/CaM engagement triggers rapid autophosphorylation at T348, which is essential for full activity. Phosphorylation on S500, by eEF-2K or PKA, lowers the CaM requirement (∼20-fold) without increasing maximal catalytic turnover. Here we show that the phosphomimetic S500D markedly enhances CaM binding in the T348-phosphorylated enzyme. S500D also elevates CaM-independent ("intrinsic") activity even in the absence of phosphorylation at T348, although maximal activity requires modification at both sites. Hydrogen-deuterium exchange mass spectrometry reveals CaM-dependent conformational changes near S500, consistent with relief of inhibitory constraints. Deletion of residues near S500 mimics S500D, increasing intrinsic activity and CaM binding in vitro and enhancing eEF-2 phosphorylation in cells, supporting an inhibitory role for this segment. Prior studies have linked S500 phosphorylation to eEF-2K degradation, suggesting a dual regulatory role. We demonstrate that phosphorylation at T348 and S500 synergize to stabilize an active-like conformation and increase CaM responsiveness, effectively lowering the Ca2+/CaM threshold for eEF-2K activation and enabling the integration of Ca2+, cAMP/PKA, and metabolic cues.

Protein kinases are enzymes that regulate many cellular events in eukaryotic cells, such as cell-cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site, as well as one or more substrate-binding site(s) that exhibit recognition features for a protein substrate. Thus, by bringing ATP and a substrate into close proximity, each protein kinase can modify its substrate by transferring the gamma phosphate of the ATP molecule to a serine, threonine, or tyrosine residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated, dependent on the phosphorylated versus dephosphorylated forms of the substrate. This unit describes an assay employing a fluorescent peptide substrate to measure the incorporation of non-radiolabeled phosphate. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore.

Intracellular sensors detect changes in levels of essential metals to initiate homeostatic responses. But, a mammalian manganese (Mn) sensor is unknown, representing a major gap in understanding of Mn homeostasis. Using human-relevant models, we recently reported that: 1) the primary homeostatic response to elevated Mn is upregulation of hypoxia-inducible factors (HIFs), which increases expression of the Mn efflux transporter SLC30A10; and 2) elevated Mn blocks the prolyl hydroxylation of HIFs by prolyl hydroxylase domain (PHD) enzymes, which otherwise targets HIFs for degradation. Thus, the mammalian mechanism for sensing elevated Mn likely relates to PHD inhibition. Moreover, 1) Mn substitutes for a catalytic iron (Fe) in PHD structures; and 2) exchangeable cellular levels of Fe and Mn are comparable. Therefore, we hypothesized that elevated Mn directly inhibits PHD by replacing its catalytic Fe. In vitro assays using catalytically active PHD2, the primary PHD isoform, revealed that Mn inhibited, and Fe supplementation rescued, PHD2 activity. However, a mutation in PHD2 (D315E) that selectively reduced Mn binding without substantially impacting Fe binding or enzymatic activity resulted in complete insensitivity of PHD2 to Mn in vitro. Additionally, hepatic cells expressing full-length PHD2 D315E were less sensitive to Mn-induced HIF activation and SLC30A10 upregulation than PHD2 wild-type . These results: 1) define a fundamental Mn sensing mechanism for controlling Mn homeostasis—elevated Mn inhibits PHD2, which functions as a Mn sensor, by outcompeting its catalytic Fe, and PHD2 inhibition activates HIF signaling to up-regulate SLC30A10; and 2) identify a unique mode of metal sensing that may have wide applicability.

p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38β dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission.

Lysyl hydroxylase 2 (LH2) catalyzes the formation of highly stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), thus promoting lung cancer metastasis through its capacity to modulate specific types of collagen cross-links within the tumor stroma. Using 1 and 2 from our previous high-throughput screening (HTS) as lead probes, we prepared a series of 1,3-diketone analogues, 1-18, and identified 12 and 13 that inhibit LH2 with IC50's of approximately 300 and 500 nM, respectively. Compounds 12 and 13 demonstrate selectivity for LH2 over LH1 and LH3. Quantum mechanics/molecular mechanics (QM/MM) modeling indicates that the selectivity of 12 and 13 may stem from noncovalent interactions like hydrogen bonding between the morpholine/piperazine rings with the LH2-specific Arg661. Treatment of 344SQ WT cells with 13 resulted in a dose-dependent reduction in their migration potential, whereas the compound did not impede the migration of the same cell line with an LH2 knockout (LH2KO).

Supplementary Materials and Methods Table S1: Data collection and refinement statistics native ALDOA and native ALDOA +TDZD8 Table S2: Data collection and refinement statistics: ALDOA-ND1 and ALDOA-ND1+TDZD-8. Table S3: Inhibition of HIF-1 activity by siRNA knockdown of glycolysis genes Table S4: Increase in glycolysis gene expression in hypoxia and the effect of siHIF-1 Table S5: Validation of screen results using alternate siRNAs Figure S1: Inhibition of HIF-1 activity and proliferation following knockdown of glycolysis genes Figure S2: Effect of knockdown of PGKs or ALDOA on HRE-luc activity on proliferation Figure S3: Effects of knockdown of ALDOA or PGK1 or PGK2 on ATP levels Figure S4: Effects of knockdown of ALDOA or PGK1 and PGK2 on HIF-1α protein levels Figure S5: Differential expression of ALDO or PGK Isoforms in tumor cells Figure S6: HIF-1 activity levels in MDA-MB-231 doxycycline inducible shALDOA clones sh8.8 and sh9.7. Figure S7: Stereo pairs of ALDOA monomer comparisons, shown as ribbon diagrams, with ligands shown as sticks Figure S8: TDZD-8 inhibition of HT-29, PANC1 and MIA PaCa-2 cancer cell proliferation, lactate formation, and HRE-luciferase reporter activity Figure S9: Effect of TDZD-8 on phospho-AMPK, phospho-p300 and ATP in PANC1 pancreatic cancer cells Figure S10: Effect of TDZD-8 treatment on levels of glycolytic intermediates and non-involved amino acids.

p38 mitogen-activated protein kinases (MAPKs) regulate early endocytic trafficking, but their effects on late endocytic trafficking remain unclear. Herein, we report that the pyridinyl imidazole p38 MAPK inhibitors, SB203580 and SB202190, induce a rapid but reversible Rab7-dependent accumulation of large cytoplasmic vacuoles. While SB203580 did not induce canonical autophagy, phosphatidylinositol 3-phosphate [PI(3)P] accumulated on vacuole membranes, and inhibition of the class III PI3-kinase (PIK3C3/VPS34) suppressed vacuolation. Ultimately, vacuolation resulted from the fusion of ER/Golgi-derived membrane vesicles with late endosomes and lysosomes (LELs), combined with an osmotic imbalance in LELs that led to severe swelling and a decrease in LEL fission. Since PIKfyve inhibitors induce a similar phenotype by preventing the conversion of PI(3)P to PI(3,5)P2, we performed in vitro kinase assays and found that PIKfyve activity was unexpectedly inhibited by SB203580 and SB202190, corresponding to losses in endogenous PI(3,5)P2 levels in treated cells. However, vacuolation was not entirely due to ‘off-target’ inhibition of PIKfyve by SB203580, as a drug-resistant p38α mutant suppressed vacuolation. Moreover, genetic deletion of both p38α and p38β rendered cells dramatically more sensitive to PIKfyve inhibitors, including YM201636 and apilimod. In subsequent ‘washout’ experiments, the rate of vacuole dissolution upon the removal of apilimod was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, p38 MAPKs act epistatically to PIKfyve to promote LEL fission; and pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs.

To identify novel inhibitors of the carbapenemase New Delhi metallo-β-lactamase (NDM) as possible therapeutic compounds, we conducted a high-throughput screen of a 43,358-compound library. One of these compounds, a 2-quinazolinone linked through a diacylhydrazine to a phenyl ring (QDP-1) (IC50 = 7.9 ± 0.5 μM), was characterized as a slow-binding reversible inhibitor (Kiapp = 4 ± 2 μM) with a noncompetitive mode of inhibition in which substrate and inhibitor enhance each other's binding affinity. These studies, along with differential scanning fluorimetry, zinc quantitation, and selectivity studies, support an allosteric mechanism of inhibition. Cotreatment with QDP-1 effectively lowers minimum inhibitory concentrations of carbapenems for a panel of resistant Escherichia coli and Klebsiella pneumoniae clinical isolates expressing NDM-1 but not for those expressing only serine carbapenemases. QDP-1 represents a novel allosteric approach for NDM drug development for potential use alone or with other NDM inhibitors to counter carbapenem resistance in enterobacterales.

SARS-CoV-2, a coronavirus strain that started a worldwide pandemic in early 2020, attaches to human cells by binding its spike (S) glycoprotein to a host receptor protein angiotensin-converting enzyme 2 (ACE2). Blocking the interaction between the S protein and ACE2 has emerged as an important strategy for preventing viral infection. We systematically developed and optimized an AlphaLISA assay to investigate binding events between ACE2 and the ectodomain of the SARS-CoV-2 S protein (S-614G: residues 1–1208 with a D614G mutation). Using S-614G permits discovering potential allosteric inhibitors that stabilize the S protein in a conformation that impedes its access to ACE2. Over 30,000 small molecules were screened in a high-throughput format for activity against S-614G and ACE2 binding using the AlphaLISA assay. A viral entry assay was used to validate hits using lentiviral particles pseudotyped with the full-length S protein of the Wuhan-1 strain. Two compounds identified in the screen, oleic acid and suramin, blocked the attachment of S-614G to ACE2 and S protein-driven cell entry into Calu-3 and ACE2-overexpressing HEK293T cells. Oleic acid inhibits S-614G binding to ACE2 far more potently than to the receptor-binding domain (RBD, residues 319–541 of SARS-CoV-2 S), potentially indicating a non-competitive mechanism. The results indicate that using the full-length ectodomain of the S protein can be important for identifying allosteric inhibitors of ACE2 binding. The approach reported here represents a rapidly adaptable format for discovering receptor binding inhibitors to S-proteins of future coronavirus strains.

Lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG, also called 2-oxoglutarate, or 2OG)-dependent oxygenase, is an endoplasmic reticulum-resident enzyme that hydroxylates telopeptidyl lysine residues on fibrillar collagen molecules. It leads to the formation of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which are more stable than lysine aldehyde-derived collagen cross-links (LCCs) generated devoid of LH2. It has been reported that LH2 enhances lung cancer metastatic and invasive proclivity and modulates the types of collagen cross-links (HLCC-to-LCC) in the tumor stroma. Herein, we prepared a series of 1,3-diketone analogs 1–18 and identified 12 and 13 that inhibit the LH2-driven hydroxylation of a collagen peptide substrate with IC50 approximately 300 nM and 500 nM, respectively. 12 and 13 demonstrate a 9-fold selectivity for LH2 over LH1 and LH3. Quantum Mechanics/Molecular Mechanics (QM/MM) modeling indicates that in addition to the relatively stronger interactions between compounds 12 and 13 with the active site, the selectivity stems from non-covalent interactions like hydrogen bonding between the morpholine/piperazine rings with LH2-specific Arg661, where the corresponding residue in LH1 and LH3 is Pro. Migration assays in the 344SQ lung adenocarcinoma cell line reveal that 13 shows anti-migration activity.

Background G protein-coupled receptor (GPCR) is the most targeted protein family by the FDA-approved drugs. GPCR-kinase 3 (GRK3) is critical for GPCR signaling. Our genomic analysis showed that GRK3 expression correlated with poor prognosis of gastric adenocarcinoma (GAC) patients. However, GRK3's functions and clinical utility in GAC progression and metastases are unknown. Methods We studied GRK3 expression in normal, primary, and metastatic GAC tissues. We identified a novel GRK3 inhibitor, LD2, through a chemical-library screen. Through genetic and pharmacologic modulations of GRK3, a series of functional and molecular studies were performed in vitro and in vivo. Impact of GRK3 on YAP1 and its targets was determined. Results GRK3 was overexpressed in GAC tissues compared to normal and was even higher in peritoneal metastases. Overexpression (OE) of GRK3 was significantly associated with shorter survival. Upregulation of GRK3 in GAC cells increased cell invasion, colony formation, and proportion of ALDH1(+) cells, while its downregulation reduced these attributes. Further, LD2 potently and specifically inhibited GRK3, but not GRK2, a very similar kinase to GRK3. LD2 highly suppressed GAC cells' malignant phenotypes in vitro. Mechanistically, GRK3 upregulated YAP1 in GAC tissues and its transcriptional downstream targets: SOX9, Birc5, Cyr61 and CTGF. Knockdown (KD) YAP1 rescued the phenotypes of GRK3 OE in GAC cells. GRK3 OE significantly increased tumor growth but LD2 inhibited tumor growth in the PDX model and dramatically suppressed peritoneal metastases induced by GRK3 OE. Conclusions GRK3, a poor prognosticator for survival, conferred aggressive phenotype. Genetic silencing of GRK3 or its inhibitor LD2 blunted GRK3-conferred malignant attributes, suggesting GRK3 as a novel therapeutic target in advanced GAC.

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds ("hits") that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2-OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111 , the "hits" were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.

Luminescence is characterized by the spontaneous emission of light resulting from either chemical or biological reactions. Because of their high sensitivity, reduced background interference, and applicability to numerous situations, luminescence-based assay strategies play an essential role in early-stage drug discovery. Newer developments in luminescence-based technologies have dramatically affected the ability of researchers to investigate molecular binding events. At the forefront of these developments are the nano bioluminescence resonance energy transfer (NanoBRET) and amplified luminescent proximity homogeneous assay (Alpha) technologies. These technologies have opened up numerous possibilities for analyzing the molecular biophysical properties of complexes in environments such as cell lysates. Moreover, NanoBRET enables the validation and quantitation of the interactions between therapeutic targets and small molecules in live cells, representing an essential benchmark for preclinical drug discovery. Both techniques involve proximity-based luminescence energy transfer, in which excited-state energy is transferred from a donor to an acceptor, where the efficiency of transfer depends on proximity. Both approaches can be applied to high-throughput compound screening in biological samples, with the NanoBRET assay providing opportunities for live-cell screening. Representative applications of both technologies for assessing physical interactions and associated challenges are discussed.

Overexpression of c-MYC protein without genomic amplification occurs in multiple cancers, including neuroblastoma and small cell lung cancer. In searching for the mechanisms of c-MYC protein overexpression, we demonstrated that the transcription factor, OCT4 mediates c-MYC transcriptional activation in progressive disease neuroblastoma. Subsequently, we identified two kinases, MAPKAPK2 (MK2) and DNA-PK, which are predicted to bind and phosphorylate OCT4 at S111 and S93 residues, respectively. Based on these novel observations, we developed a cell-based luminescence assay to screen and identify compounds that inhibit the interactions between MK2 and OCT4. By screening 79,671 compounds, we identified 65 compounds we designated as “hits”. Using a two-step validation of co-immunoprecipitation and pOCT4S111 detection, the compounds were further narrowed down to three for further studies. The three compounds were tested for their ability to inhibit kinase activity, in vitro cytotoxic activity, and anti-inflammatory activity. In conclusion, we developed a cell-based luminescence assay for the discovery of new agents targeting the c-MYC transcriptional activation pathway. Screening and subsequent validation identified a small number of compounds for further development.

Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the S111 and the S93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between DNA-PKcs and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds (“hits”) that inhibited the luminescence reaction. Using a custom antibody specific for pOCT4S93, the “hits” were validated for their effect on OCT4 phosphorylation and activation. Seven compounds were selected for the second step of validation, which focused on the interaction between kinase and substrate. After further characterization, we identified two compounds that significantly impaired the ability of DNA-PKcs to bind to and phosphorylate OCT4. The compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found two compounds that may function as lead compounds for further development.