High-throughput Biochemical Assay Research Articles

Abstract C075: A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade

Abstract Background: SHP1 is a member of the src homology 2 (SH2) domain-containing protein phosphatases. It contains two SH2 domains at the N-terminus, a catalytic domain and a C-terminal tail. SHP1 is primarily expressed in hematopoietic cells and commonly recognized as a negative regulator of multiple signaling pathways. It has been known that SHP1 interacts with proteins of the inhibitory-receptor superfamily containing immunoreceptor tyrosine-based inhibitory motifs. Methods: To screen for SHP1 allosteric inhibitors, we employed a DiFMUP high-throughput biochemical assay system. Compounds that only perturbed the activity of SHP1 was further optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. X-ray crystallography has been introduced to determine the interaction between the inhibitor and the allosteric site of SHP1. We also evaluated the lead series in in-vitro studies with human primary immune cells and in-vivo efficacy studies in syngeneic mouse models. Results: We discovered a series of SHP1 allosteric inhibitors with IC50 values at the nanomolar level and high selectivity over the other PTP enzymes, including SHP2. Structurally, SHP1 adopts an autoinhibited conformation in its basal state, where the N-SH2 domain interacts with the PTP domain and blocks access to the catalytic site. X-ray crystallography reveals that our SHP1 inhibitors are bound to non-catalytic sites of the SHP1 inactive conformation. Among the compounds, SB6299 demonstrated potent cytokine responses in various human primary immune cell types. SB6299 significantly enhanced IL-2, IFN-γ, and TNF-α productions in human primary pan-T, NK, and monocyte cells, respectively. The cytokine release was confirmed as a result of selective SHP1 inhibition through the evaluation of SHP1 knockout in primary immune cells. Moreover, SB6299 showed dose responses in costimulatory molecules and cytokines (TNF-α and IL6) in human monocyte derived dendritic cells (DCs) and enhanced DC maturation markers such as CD80, CD86, and CD40. The anti-tumor efficacy of SB6299 was evaluated in the CT26 and MC38 syngeneic mouse models. Orally administered SB6299 showed tumor growth inhibition as a single agent, and the efficacy was further enhanced when administered in combination with an anti-PD1 antibody. Conclusions: 1. Our studies successfully demonstrate that SHP1 can be allosterically inhibited, allowing selectivity with other phosphatases, including SHP2. 2. SB6299 is the first-in-class orally available, potent, and selective SHP1 allosteric inhibitor. 3. Attenuation of SHP1 activity by SB6299 could promote cytokine secretion and cytotoxic effects in human primary immune cells. 4. Orally administered SB6299 induced tumor growth inhibition as a single agent and in combination with an anti-PD1 antibody. 5. Currently, more selective and potent inhibitors are being tested. Citation Format: Jun Gyu Kim, Ki Moon Ryu, Mi Yeon Jang, Jongho Cho, Dajeong Kim, Dae Hyeon Seong, Kyu Hwan Kim, Ik-Soo Jang, Jeong Yoon Shin, Hong Sik Han, Joon Yonug Hwang, Dae Young Lee, Chae Lim Ryu, Song Yi Lee, Tae-Dong Han, SeungMin Yang, Hyounmie Doh. A first-in-class and highly selective SHP1 allosteric inhibitor exhibits robust anti-tumor immunity and synergizes with PD-1 blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C075.

Abstract 6181: Cell line panel with HIBIT tagged endogenous proteins to accelerate PROTAC drug discovery

Abstract Recent years have seen great advancement of targeted protein degradation technology, in particular, proteolysis targeting chimeras (PROTACs) are now widely used in developing therapeutics for treating cancer. A PROTAC is a heterobifunctional small molecule with three distinct moieties: a ligand to bind a targeted protein of interest (POI), a second ligand to recruit E3 ubiquitin ligase to form a ternary complex, and a linker for bridging the two ligands. Through the E3 ubiquitin ligase pathway, properly designed PROTACs can effectively degrade POIs with high specificity, which can be pathogenic proteins, thus regulate related pathways and inhibit tumor growth. Currently, there is no high-throughput biochemical assay to measure the POI degradation in vitro, hindering the application of the PROTAC technology. Here we report the development of a high-throughput cell based assay to quantitatively measure the endogenous drug target degradation induced by PROTACs. This assay encompasses over 800 cancer cell lines, many of which are CRISPR-engineered ones on common drug targets. A unique feature of our assay is the incorporation of HiBiT short sequence tags on either N-terminal or C-terminal of endogenous protein in E3 ligase matched cell line through site-specific gene homologous recombination technology. By highly specific and sensitive detection of HiBiT tag content in cell lysates with biochemical assay, we can determine target protein degradation by PROTAC treatment. We demonstrate the utility of the assay on a wide array of drug targets including RAS, LDHA, BTK and HPK1, and show that it can accelerate PROTAC drug discovery. Citation Format: Yunpeng Zhai, Ming Tan, Defu Liu, Guoqian Wang, Jinying Ning, Feng Hao. Cell line panel with HIBIT tagged endogenous proteins to accelerate PROTAC drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6181.

Benzilydene and thiourea derivatives as new classes of carbonic anhydrase inhibitors: an in vitro and molecular docking study

Carbonic anhydrase-II (CA-II) catalyzes reversible hydration of carbon dioxide leading to the formation of bicarbonate and a proton. CA-II inhibitors act as biomarkers and therapeutic targets for various diseases such as epilepsy, glaucoma, leukemia, and cystic fibrosis. In the present work, a number of thiourea and benzylidene derivatives were evaluated as CA-II inhibitors. For this, detection of CA-II inhibition was tested through high throughput biochemical mechanism-based assay. Results indicated that thiourea derivatives exhibit remarkable inhibition activity with IC50 values ranging from 1.90 ± 1.30 to 25.90 ± 2.05 µM, when compared with the standard inhibitor acetazolamide IC50 = 0.13 ± 0.05. On the other hand, bezylidene derivatives showed weak inhibition potential with IC50 values in the range of 52.68 ± 0.47 to 348.57 ± 3.32 µM when compared with the thiourea derivatives; the Ez-Fit enzyme kinetics software was used for IC50 calculations from the percentage inhibition. In addition, 3T3 normal cell line was employed to determine the toxicity profile of identified inhibitors of CA-II. In silico modeling was performed to decipher the molecular nature of thiourea and benzylidene derivatives with key residues of carbonic anhydrase; results were in agreement with experiments related to inhibition of carbonic anhydrase.

Identification of Novel Trypanosoma cruzi Proteasome Inhibitors Using a Luminescence-Based High-Throughput Screening Assay.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening condition that has become a global issue. Current treatment is limited to two medicines that require prolonged dosing and are associated with multiple side effects, which often lead to treatment discontinuation and failure. One way to address these shortcomings is through target-based drug discovery on validated T. cruzi protein targets. One such target is the proteasome, which plays a crucial role in protein degradation and turnover through chymotrypsin-, trypsin-, and caspase-like catalytic activities. In order to initiate a proteasome drug discovery program, we isolated proteasomes from T. cruzi epimastigotes and characterized their activity using a commercially available glow-like luminescence-based assay. We developed a high-throughput biochemical assay for the chymotrypsin-like activity of the T. cruzi proteasome, which was found to be sensitive, specific, and robust but prone to luminescence technology interference. To mitigate this, we also developed a counterscreen assay that identifies potential interferers at the levels of both the luciferase enzyme reporter and the mechanism responsible for a glow-like response. Interestingly, we also found that the peptide substrate for chymotrypsin-like proteasome activity was not specific and was likely partially turned over by other catalytic sites of the protein. Finally, we utilized these biochemical tools to screen 18,098 compounds, exploring diverse drug-like chemical space, which allowed us to identify 39 hits that were active in the primary screening assay and inactive in the counterscreen assay.

Development of a High-Throughput Biochemical Assay to Screen for Inhibitors of Aerobactin Synthetase IucA

Acquiring sufficient quantities of iron to support survival is often a critical limitation for pathogenic bacteria. To meet this demand, bacteria have evolved unique strategies to scavenge iron and circumvent the nutritional immunity exerted by their hosts. One common strategy, which is often a key virulence factor for bacterial pathogens, involves the synthesis, secretion, and reuptake of iron chelators known as siderophores. In vitro and in vivo studies have demonstrated that the siderophore aerobactin is critical for virulence in the hypervirulent pathotype of Klebsiella pneumoniae (hvKP). Given the high rate of multidrug resistance in K. pneumoniae, and in light of the ever-increasing demand for novel Gram-negative therapeutic targets, we identified aerobactin production as a promising antivirulence target in hvKP. Herein, we describe the development of a high-throughput biochemical assay for identifying inhibitors of the aerobactin synthetase IucA. The assay was employed to screen ~110,000 compounds across several commercially available small-molecule libraries. IucA inhibitors with activity at micromolar concentrations were identified in our screening campaigns and confirmed using secondary orthogonal assays. However, the most potent compounds also exhibited some properties commonly observed with promiscuous/nonspecific inhibitors, including incubation time and target enzyme concentration dependence, as well as the potential to antagonize unrelated enzymes.

Pharmacological Validation of N-Myristoyltransferase as a Drug Target in Leishmania donovani

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for ∼30 000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, Ki of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC50 of 2.4 μM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.

Abstract 3022: Novel hydrocarbon stapled peptide inhibitors suppresses β-catenin dependent cancer growth and immunomodulates colorectal tumors

Abstract Mutations in several components of the canonical Wnt pathway function as drivers in a range of common solid tumor cancers, including about 90% of sporadic colorectal cancers (CRCs), the third most common cancer in the US. In particular, activation of β-catenin (β-cat), a key Wnt signaling component, can initiate tumorigenesis in the intestine and promote cancer stem cell metastasis6,7. As such, targeting the Wnt pathway is an attractive approach to the development of cancer therapeutics. However, Wnt inhibitors tested to date, including those that disrupt β-cat activity, have either had limited efficacy or unacceptable side effect profiles1. Here, we developed a potent and robust, selective lead compound targeting oncogenic Wnt activity by disrupting the highly specific interaction of β-cat with its co-activators BCL9/B9L, which has been shown to abrogate transactivation. By using hydrocarbon stapled peptide technology and an acutely sensitive high throughput biochemical screening assay, we have identified several promising peptides that possess a strong pharmacokinetic profile. These peptides (i) exhibit robust activity in mouse models; (ii) inhibit cancer cell proliferation, metastasis, and stem cell progression; and (iii) modulate the immune microenvironment, demonstrating the feasibility of such peptide development and selection. Synergy is demonstrated between the stapled peptides and anti-PD1 antibodies, which has significant implications for combination therapy with commercial immune checkpoint blockers that have become standard of care for second-line melanoma treatment. These peptides represent a promising new potential therapeutic class for patients with CRC, for which traditional chemotherapy and targeted inhibitors have had limited therapeutic success. Citation Format: David Zhu, Joy Jin. Novel hydrocarbon stapled peptide inhibitors suppresses β-catenin dependent cancer growth and immunomodulates colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3022. doi:10.1158/1538-7445.AM2017-3022

Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

A Novel High Throughput Biochemical Assay to Evaluate the HuR Protein-RNA Complex Formation

The RNA binding protein HuR/ELAVL1 binds to AU-rich elements (AREs) promoting the stabilization and translation of a number of mRNAs into the cytoplasm, dictating their fate. We applied the AlphaScreen technology using purified human HuR protein, expressed in a mammalian cell-based system, to characterize in vitro its binding performance towards a ssRNA probe whose sequence corresponds to the are present in TNFα 3’ untranslated region. We optimized the method to titrate ligands and analyzed the kinetic in saturation binding and time course experiments, including competition assays. The method revealed to be a successful tool for determination of HuR binding kinetic parameters in the nanomolar range, with calculated Kd of 2.5±0.60 nM, k on of 2.76±0.56*106 M-1 min-1, and k off of 0.007±0.005 min-1. We also tested the HuR-RNA complex formation by fluorescent probe-based RNA-EMSA. Moreover, in a 384-well plate format we obtained a Z-factor of 0.84 and an averaged coefficient of variation between controls of 8%, indicating that this biochemical assay fulfills criteria of robustness for a targeted screening approach. After a screening with 2000 small molecules and secondary verification with RNA-EMSA we identified mitoxantrone as an interfering compound with rHuR and TNFα probe complex formation. Notably, this tool has a large versatility and could be applied to other RNA Binding Proteins recognizing different RNA, DNA, or protein species. In addition, it opens new perspectives in the identification of small-molecule modulators of RNA binding proteins activity.

Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the "RIP-Chip" assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.

A homogeneous, solid-phase assay for hepatitis C virus RNA-dependent RNA polymerase

Discovery and development of effective antiviral agents to combat hepatitis C virus (HCV) is the focus of intensive research both in academia and in pharmaceutical companies. One of the HCV nonstructural proteins, NS5B (an RNA-dependent RNA polymerase), represents an attractive target in light of the clinical success of human immunodeficiency virus reverse transcriptase inhibitors. To identify and evaluate NS5B inhibitors, we developed a homogeneous, solid-phase, high-throughput biochemical assay for detecting NS5B enzymatic activity. In this assay, a biotinylated oligo(dT 12) primer was immobilized onto streptavidin-coated scintillation proximity assay (SPA) beads, and after addition of homopolymeric A template, NS5B enzyme, and radiolabeled uridine 5′-triphosphate, the primer-dependent RNA synthesis occurred on beads. Optimization of the on-bead reaction resulted in the use of significantly less RNA template and NS5B enzyme while producing a faster steady state reaction rate compared to the solution-phase or off-bead SPA. The newly developed solid-phase assay is functionally comparable to the solution-phase assay as similar potencies of HCV NS5B inhibitors tested were obtained with the two assays. Furthermore, the solid-phase assay offers the advantage of delaying initiation, mimicking a physical preincubation step required for evaluating time-dependent inhibitors.

Clavaric acid and steroidal analogues as Ras- and FPP-directed inhibitors of human farnesyl-protein transferase.

We have identified a novel fungal metabolite that is an inhibitor of human farnesyl-protein transferase (FPTase) by randomly screening natural product extracts using a high-throughput biochemical assay. Clavaric acid [24, 25-dihydroxy-2-(3-hydroxy-3-methylglutaryl)lanostan-3-one] was isolated from Clavariadelphus truncatus; it specifically inhibits human FPTase (IC50 = 1.3 microM) and does not inhibit geranylgeranyl-protein transferase-I (GGPTase-I) or squalene synthase activity. It is competitive with respect to Ras and is a reversible inhibitor of FPTase. An alkaline hydrolysis product of clavaric acid, clavarinone [2,24,25-trihydroxylanostan-3-one], lacking the 3-hydroxy-3-methylglutaric acid side chain is less active as a FPTase inhibitor. Similarly, a methyl ester derivative of clavaric acid is also inactive. In Rat1 ras-transformed cells clavaric acid and lovastatin inhibited Ras processing without being overtly cytotoxic. Excess mevalonate reversed the effects of lovastatin but not of clavaric acid suggesting that the block on Ras processing by clavaric acid was due to inhibition of FPTase and not due to inhibition of HMG-CoA reductase. Despite these results, the possibility existed that clavaric acid inhibited Ras processing by directly inhibiting HMG-CoA reductase. To directly examine the effects of clavaric acid and clavarinone on HMG-CoA reductase, cholesterol synthesis was measured in HepG2 cells. No inhibition of HMG-CoA reductase was observed indicating that the inhibition of Ras processing by this class of compounds is due to inhibition of FPTase. To date, clavaric acid is the second reported nitrogen-free compound that competes with Ras to inhibit FPTase activity. A series of related compounds derived from computer-based similarity searches and subsequent rational chemical synthetic design provided compounds that exhibited a range of activity (0.04 --> 100 microM) against FPTase. Modest changes in the structures of these inhibitors dramatically change the inhibitory activity of these inhibitors.

A High-Throughput STAT Binding Assay Using Fluorescence Polarization

STAT (signal transducers and activators of transcription) is a class of transcription factors that are activated upon cytokine or growth factor binding to cell surface receptors. Activated STAT proteins dimerize, translocate into the nucleus, and activate transcription, leading to various immune responses. The inhibition of STAT binding to cell receptors might provide a means to modulate these immune responses. We developed a high-throughput biochemical assay to measure the interaction between an interferon-γ receptor-derived phosphotyrosine-containing peptide and STAT1, using fluorescence polarization as the detection method. This assay can be used to screen for small molecules capable of disrupting receptor–STAT interactions.