Evaluation Of Small Molecule Inhibitors Research Articles

Abstract 6584: Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer

Abstract While checkpoint inhibitor treatment has extended the survival of many cancer patients, most initial responders will later develop disease progression through a variety of poorly understood acquired resistance mechanisms (CPI-AR). One such recently described mechanism involves the transcriptional hyperactivation of genes associated with interferon (IFN) signaling, JAK/STAT, and antigen processing/presentation pathways. Aberrant IFN regulation in the setting of AR suggests that tumor cell intrinsic mechanisms have conferred a growth advantage despite the presence of immune-mediated pressure unleashed by PD-1/L1 antibody blockade. Analysis of transcripts progressively upregulated during acquisition of AR in vivo and transcripts upregulated by IFN exposure of isolated AR cell lines in vitro identified the E3 ubiquitin ligase TRIM7 as a candidate mediator of the AR phenotype. A specific pocket on the PRY/SPRY domain of TRIM7 has been shown to bind viral peptides, resulting in ubiquitination and degradation as a mechanism to mitigate viral propagation. Here we describe the evaluation of small molecule inhibitors (SMI) designed to the TRIM7 PRY/SPRY viral-pocket domain, where binding specificity was confirmed using mass spec analysis. These SMI competitively inhibited TRIM7 mediated stabilization of RACO-1, resulting in decreased cancer cell proliferation which was associated with altered c-Jun/AP-1 transcription. TRIM7 SMI also disrupted STING and MAVS ubiquitination and restored IFN responsiveness and sensitivity to PD-1 inhibitor treatment. Finally, given the position of TRIM7 downstream of growth factor receptor and kras signaling, combination of TRIM7 inhibitors with kras, braf, and/or MEK inhibitors was shown to additively suppress proliferation of tumor cells with kras mutations. These results provide evidence that upregulation of TRIM7 mediates both a pro-proliferative and IFN resistant phenotype in the setting of CPI-AR, and that inhibition of TRIM7 with small molecule inhibitors may prevent or reverse AR to PD-1/L1 blockade. Citation Format: Thuy-Ai Tran Nguyen, Marc Morra, Kevin Li, Ulhas Bhatt, Joseph Mwangi, Yisel Rivera-Molina, Nathan Oien, Julio Medina, Taylor H. Schreiber, George Fromm. Aberrant TRIM7 expression potentiates RACO-1 mediated proliferation and dysregulated interferon responsiveness in the setting of anti-PD-1 acquired resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6584.

Synthesis and Evaluation of Small Molecule Inhibitors of the Androgen Receptor N-Terminal Domain.

The androgen receptor (AR) is central to prostate cancer pathogenesis and has been extensively validated as a drug target. However, small-molecule anti-androgen therapies remain limited due to resistance and will eventually fail to suppress tumor growth, resulting in progression to castration-resistant prostate cancer (CRPC). The intrinsically disordered N-terminal domain (NTD) is crucial for AR transactivation and has been investigated as a suitable target in the presence of ligand binding domain mutations. A screening campaign identified biaryl isoxazole compound 7 as a weak inhibitor of the AR NTD. A library of biaryl analogues were synthesized, and their biological activities were assessed in a VCaP cell-based luciferase reporter gene assay. A structure-activity relationship (SAR) study revealed that indazole analogue 16 exhibited increased potency and favorable physicochemical properties with a benchmarked pharmacokinetic profile, providing a suitable starting point for further optimization of 16 as a CRPC therapeutic in the presence of AR mutations.

Abstract 461: Targeting glucose metabolism in lung cancer

Abstract Positron emission tomographic studies with 2-18F-fluoro-deoxy-glucose (FDG-PET) consistently demonstrate that non-small cell lung cancers (NSCLC) take up significantly more glucose than normal lung. NSCLCs preferentially utilize glucose to support their rapid growth- a phenotype driven by several oncogenic alterations among which mutations of the oncoprotein Ras are frequently observed. Oncogenic Ras is shown to activate downstream effectors (e.g. HIF-1α) that stimulate glycolysis - in part by activation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family of enzymes (PFKFB1-4). The PFKFBs produce fructose-2,6-bisphosphate (F26BP) which activates a key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK1). In preliminary studies, we found that the PFKFB4 family member was highly expressed relative to other PFKFB isoforms (by multiplex RT-PCR) in a series of patient-derived NSCLCs relative to paired normal lung tissue. We further explored PFKFB4 protein expression by immunohistochemistry in NSCLCs derived from a cohort of patients and found markedly elevated PFKFB4 in the majority of tumors relative to adjacent normal tissue. We then determined that silencing PFKFB4 markedly reduced F26BP, glycolysis and cell survival in NSCLC cell lines and inhibited xenograft lung tumor growth in vivo. In order to target PFKFB4, we conducted a virtual screen of compounds that fit into the active site of PFKFB4 and discovered a first-in-class small molecule selective PFKFB4 inhibitor, termed 5MPN, that selectively inhibits recombinant PFKFB4 activity and decreases F26BP, glycolysis and proliferation in multiple NSCLC cell lines, with significantly higher anti-proliferative effects in Ras-driven cell lines. 5MPN also caused marked suppression of growth in established NSCLC xenograft tumors, importantly without systemic toxicity. We have developed an inducible, pan-tissue homozygous floxed Pfkfb4 knockout mouse and crossed it with a Cre recombinase mouse driven by a tamoxifen-inducible β-actin promoter to produce Cre/Pfkfb4fl/fl mice. We crossed Cre/Pfkfb4fl/fl mice with a Ras-driven lung cancer model, induced Pfkfb4 deletion by tamoxifen administration and found significantly lower total lung tumor burden in these mice accompanied by decreased PFKFB4 expression in tumors and lung tissue. Taken together, these data provide strong rationale for the further evaluation of small molecule inhibitors of PFKFB4 as a potential therapeutic strategy against NSCLC. Citation Format: Sucheta Telang, Simone Chang, Nicole Sanders. Targeting glucose metabolism in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 461.

Synthesis and evaluation of small molecule inhibitors of LSD1 for use against MYCN-expressing neuroblastoma

The epigenetic regulator lysine specific demethylase 1 (LSD1), a MYCN cofactor, cooperatively silences MYCN suppressor genes. Furthermore, LSD1 has been correlated with adverse effects in neuroblastic tumors by propagating an undifferentiated, malignant phenotype. We observed that high LSD1 mRNA expression in MYCN-expressing neuroblastoma (NB) correlated with poor prognosis, implicating LSD1 as an oncogenic accomplice in high-grade NB. Thus, LSD1 inhibition is a potential strategy for targeting treatment-resistant MYCN-expressing NB. Tranylcypromine-based covalent LSD1 inhibitors have demonstrated anti-tumor activity but are associated with undesirable off-target effects, such that only 2 non-covalent LSD1 inhibitors are in clinical trials. We now report 3 novel scaffolds for reversible LSD1 inhibition: 2-(arylsulfonamido)benzoic acid, N-(2-(1H-tetrazol-5-yl)phenyl)benzenesulfonamide and 2-(arylcarboxamido)benzoic acid analogues. The most active of these analogues, compound 48, exhibited potent and selective mixed reversible inhibition of LSD1 (IC50 = 0.58 μM) and significantly increased global H3K4me2 in NB cells. In addition, combination treatment with 48 and bortezomib in NB cells results in a synergistic effect.

Evaluation of Small Molecule Inhibitors of Pseudomonas Virulence factor LasB as Non‐Traditional Immunotherapeutics

Pseudomonas aeruginosa can cause severe opportunistic pulmonary infections associated with mechanical ventilation and the genetic disease cystic fibrosis. Due to its multidrug resistance, it is a priority pathogen for therapeutic development. Inflammation during such infections, which causes destruction of lung architecture and function, is activated in part through the maturation of IL‐1β by the virulence factor LasB. Previous results in mouse models suggest that matrix metalloprotease inhibitor drugs also inhibit LasB, preventing cell death and inflammation caused by caused by P. aeruginosa. We hypothesized that other inhibitors of matrix metalloproteases, including tetracycline‐family antibiotics, could be used to target LasB. We evaluated small molecule inhibitors against LasB activation during infections of macrophages, neutrophils, and monocytes and the synergy of these inhibitors with conventional antibiotics and anti‐inflammatories. We first optimized the drug concentration by testing the ability of the various drugs to inhibit LasB at various dilutions. Using an effective concentration, we then infected the various cell types with P. aeruginosa and analyzed the impact of the inhibitors on cell death and maturation of IL‐1β in infected cells. The results indicate that several tetracycline‐family compounds inhibit LasB during P. aeruginosa infections and decrease cell death. This was true for compounds that had no antimicrobial activity yet still inhibited LasB. In addition, the results suggest that the drugs target different cell types. Together, we find that tetracyclines may have therapeutic benefits, even against resistant bacteria, due to their ability to decrease inflammation. This may serve as an effective adjunctive therapy during infection to limit tissue damage and give the immune system and antibiotics more time to respond.

Comparison of Cellular Target Engagement Methods for the Tubulin Deacetylases Sirt2 and HDAC6: NanoBRET, CETSA, Tubulin Acetylation, and PROTACs.

The tubulin deacetylases Sirt2 and HDAC6 have been associated with the development of various diseases. Herein, we discuss recent approaches that enable cellular target engagement studies for these deacetylases and thus play a critical role in the evaluation of small molecule inhibitors of Sirt2 or HDAC6 as potential therapeutic agents.

Naphthalene Monoimide Derivative Ameliorates Amyloid Burden and Cognitive Decline in a Transgenic Mouse Model of Alzheimer's Disease

AbstractAlzheimer's disease (AD) is a major neurodegenerative disorder and the leading cause of dementia worldwide. Predominantly, misfolding and aggregation of amyloid‐β (Aβ) peptides associated with multifaceted toxicity is the neuropathological hallmark of AD pathogenesis and, thus the primary therapeutic target to ameliorate neuronal toxicity and cognitive deficits. Herein, the design, synthesis, and evaluation of small molecule inhibitors with naphthalene monoimide scaffold to ameliorate in vitro and in vivo amyloid induced neurotoxicity are reported. The detailed studies establish TGR63 as the lead candidate to rescue neuronal cells from amyloid toxicity. The in silico studies show the disruption of salt bridges and intermolecular hydrogen bonding interactions within Aβ42 fibrils by the interaction of TGR63, causing destabilization of Aβ42 assembly. Remarkably, TGR63 treatment shows a significant reduction in cortical and hippocampal amyloid burden in the progressive stages of APP/PS1 AD mice brain. Various behavioral tests demonstrate rescued cognitive deficits. The excellent biocompatibility, blood–brain barrier permeability, and therapeutic efficacy to reduce the amyloid burden make TGR63 a promising candidate for the treatment of AD.

A direct fluorometric activity assay for lipid kinases and phosphatases

Lipid kinases and phosphatases play key roles in cell signaling and regulation, are implicated in many human diseases, and are thus attractive targets for drug development. Currently, no direct in vitro activity assay is available for these important enzymes, which hampers mechanistic studies as well as high-throughput screening of small molecule modulators. Here, we report a highly sensitive and quantitative assay employing a ratiometric fluorescence sensor that directly and specifically monitors the real-time concentration change of a single lipid species. Because of its modular design, the assay system can be applied to a wide variety of lipid kinases and phosphatases, including class I phosphoinositide 3-kinase (PI3K) and phosphatase and tensin homolog (PTEN). When applied to PI3K, the assay provided detailed mechanistic information about the product inhibition and substrate acyl-chain selectivity of PI3K and enabled rapid evaluation of small molecule inhibitors. We also used this assay to quantitatively determine the substrate specificity of PTEN, providing new insight into its physiological function. In summary, we have developed a fluorescence-based real-time assay for PI3K and PTEN that we anticipate could be adapted to measure the activities of other lipid kinases and phosphatases with high sensitivity and accuracy.

Symmetry-based ligand design and evaluation of small molecule inhibitors of programmed cell death-1/programmed death-ligand 1 interaction.

The development of small molecule inhibitors of PD-1/PD-L1 is eagerly anticipated for treatment of cancer. We focused on the symmetry of the ternary complex structure of reported small molecule ligands and hPD-L1 homodimers, and designed partially- or fully-symmetric compounds for more potent inhibitors. The design of the new compounds was guided by our hypothesis that the designed symmetric compound would induce a flip of sidechain of ATyr56 protein residue to form a new cavity. The designed compound 4 exhibited substantially increased binding affinity to hPD-L1, as well as PD-1/PD-L1 inhibitory activity in physiological conditions. Compound 4 also showed a dose-dependent increase in IFN-γ secretion levels in a mixed lymphocyte reaction assay. These results not only indicate the feasibility of targeting the PD-1/PD-L1 pathway with small molecules, but illustrate the applicability of the symmetry-based ligand design as an attractive methodology for targeting protein-protein interaction stabilizers.

Biochemical Characterization of In vitro Reconstituted Biologically Active Recombinant Shiga Toxin.

Shiga toxins comprise a family of related proteins produced by bacteria Shigella dysenteriae and some strains of Escherichia coli that cause severe clinical manifestations. Severe Shiga toxin intoxication results in Haemolytic-Uremic Syndrome (HUS), up to 50% of HUS patients manifest some degree of renal failure and ~10% of such cases develop permanent renal failure or death. In present research work production of biologically active rStx from non-toxic rStxA and rStxB subunits were established that can be used in many biomedical applications. Purification of Shiga toxin from bacteria is a multistep time consuming process resulting in low yield. To overcome this problem, the rStxA and rStxB protein were separately cloned and expressed in E. coli host and purified through affinity chromatography. GST pull-down assay was performed for interaction study between rStxA and pentameric rStxB. The affinity between A and B subunits of reconstituted recombinant Shiga toxin (AB5) was determined by SPR. The biological activity of the toxin was confirmed in Vero cells and mouse lethality assay. The yield of GST-StxA and His6X-StxB obtained after affinity chromatography was estimated to 2 and 5 mg/l, respectively. Samples analyzed in pull down assay revealed two bands of ~58 kDa (rStxA) and ~7.7 kDa (rStxB) on SDS-PAGE. Affinity was confirmed through SPR with KD of 0.85 pM. This rStx produced from 1:5 molar ratio found to be cytotoxic in Vero cell line and resulted lethality in mouse. Large scale production of rStx using the method can facilitate screening and evaluation of small molecule inhibitors for therapeutics development.

Novel Cell Model for Tauopathy Induced by a Cell-Permeable Tau-Related Peptide.

In the present study, a cell penetrating peptide (CPP)-amyloid conjugate was prepared (T-peptide), where the amyloid-forming sequence was homologous to a nucleating sequence from human Tau protein (306VQIVYK311). Kinetic and biophysical studies showed the peptide formed long-lived oligomers which were taken up by endocytosis and localized in perinuclear vesicles and in the cytoplasm of murine hippocampal neuroblastoma cells and human HeLa cells. Thioflavin S (ThS) staining of amyloid colocalized with pathological phosphorylated Tau, suggesting that the peptide was able to seed endogenous wild-type Tau. Subsequent experiments showed that aggregates present in the lysosomes mediated lysosome membrane permeability (LMP). We observed a decrease in total Tau, irrespective of phosphorylation state, consistent with Tau fragmentation by lysosomal proteases. We found cytotoxicity of T-peptide could be abrogated by inhibitors of lysosomal hydrolases and caspases, consistent with a model where Tau fragments processed by the lysosome leak into the cytoplasm and induce toxicity in subsequent downstream steps. It is our hope that the T-peptide system may prove amenable to the evaluation of small molecule inhibitors of cytotoxicity, especially those which target either Tau aggregation or the lysosomal/autophagy system.

PAIN-less identification and evaluation of small molecule inhibitors against protein tyrosine phosphatase 1B.

A highly miniaturized biochemical assay was set up to test a focused set of natural products against the enzymatic activity of protein tyrosine phosphatase 1B (PTP1B). The screen resulted in the identification of the natural product alkaloids, berberine and palmatine as well as α-tocopheryl succinate (α-TOS) as potential inhibitors of PTP1B. In a second step, several read-out and counter assays were applied to confirm the observed inhibitory activity of the identified hits and to remove false positives which target the enzymatic activity of PTP1B by a non-specific mechanism, also known as PAINS (pan-assay interference compounds). Both, berberine and palmatine were identified as false positives which interfered with the assay read-out. Using NMR spectroscopy, self-association via stacking interactions was detected for berberine in aqueous media, which may also contribute to the non-specific inhibition of PTP1B. α-TOS was confirmed as a novel reversible and competitive inhibitor of PTP1B. A concise structure-activity relationship study identified the carboxyl group and the saturated phytyl-side chain as being critical for PTP1B inhibition.

Design, Synthesis and Biological Evaluation of Small-Molecule Inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) Signaling Pathway

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is involved in the occurrence and development of the tumors, and is regarded as an attractive therapeutic target for cancer therapy. Our laboratory discovered some STAT3 inhibitors containing benzothiazole scaffold through virtual screening before. In a continuing effort to develop more potential STAT3 inhibitors, twenty-one target compounds based on our identified hit compound (16v) were rational designed and synthesized. These structures were characterized by H NMR, C NMR and HRMS. All the target compounds were tested for their inhibitory activity using a STAT3 luciferase reporter system. The results showed that many compounds displayed better activity than lead compound 16v in series I. However, compounds containing thiazolo[5,4-d]pyrimidine scaffold led to the loss of inhibitory activity. This may attributed to the losing of hydrogen bonding to Glu638.

In vitro Evaluation of Small Molecule Inhibitors and Probiotic Byproducts on Growth and Viability of Vaginal Microorganisms

Background: Interest in avoidance of antibiotic resistance development through nonantibiotic therapeutics prompts development of topical vaginal preparations that may Original Research Article British Journal of Medicine & Medical Research, 4(36): 5779-5792, 2014 5780 inhibit vaginal microorganisms. Prior data on boric acid, zinc salts and probiotic microorganisms suggests a potential role in female genital tract infections. Aims: Determine antimicrobial effects of boric acid, zinc sulfate and metabolites of probiotic lactobacilli alone or in combination on Candida albicans, Gardnerella vaginalis and Streptococcus agalactiae. Methodology: Absolute counts of test organisms cultivated with or without inhibitory compounds were determined by a flow cytometric method and compromise of microbial cell integrity was demonstrated by propidium iodide staining, also determined by flow cytometry. All microbial count growth experiments were conducted in triplicate and averages were reported. Results: The three microbial species challenged with boric acid, zinc sulfate or spent medium from probiotic lactobacilli showed varying degrees of susceptibility to these inhibitors, with boric acid showing consistent activity against C. albicans and also showing yeast cell damage demonstrated by propidium iodide uptake. Boric acid showed greater antibacterial activity against S. agalactiae than against G. vaginalis and neither of the bacterial organisms showed propidium iodide staining. Zinc sulfate inhibition was greatest for Candida and least for Gardnerella. Probiotic Lactobacillus spent media was also inhibitory toward the three test organisms with Gardnerella being slightly less susceptible than the other two test organisms. When binary combinations were tested, the combination of boric acid and Lactobacillus spent medium was the most effective in vitro against all three organisms. Conclusion: Zinc sulfate did not prove any more effective in vitro against the three test organisms than did boric acid or Lactobacillus spent media. The most potent binary combination against the three test microorganisms was boric acid plus spent media from probiotic Lactobacilli.

Abstract 2470: Identification and evaluation of small molecule inhibitors of lysophosphatidic acid acyltransferase-beta.

Abstract Abstract: Lysophosphatidic acid acyltransferase-beta (LPAAT-β) catalyzes the production of phosphatidic acid (PA) from lysophosphatidic acid (LPA). PA is a lipid cofactor that contributes to the activation of c-Raf, BRAF, mTOR and PKC-ζ. LPAAT-β expression is a prognostic factor in gynecologic malignancies and is being investigated as a therapeutic target in a variety of tumor types. We previously reported that knockdown of LPAAT-β by siRNA led to a significant inhibition of both anchorage-dependent and -independent growth in pancreatic cancer cell lines. Having thus established LPAAT-β as a valid therapeutic target in pancreatic cancer, we screened small molecule libraries for compounds which block LPAAT-β enzymatic activity in vitro. Methods: We utilized a 96-well plate screening assay for LPAAT-β enzymatic activity modified from Aguado and Campbell, J Biol Chem 273(7):4096-4105, 1998. Briefly, compounds were introduced into a mixture containing full-length, recombinant, baculovirus-produced LPAAT-β (Blue Sky Biotechnology), oleoyl-LPA, and oleoyl coenzyme A. The ability of the compound to inhibit the enzyme activity was assayed by the measurement of the reaction of the thiol group of the released coenzyme A with the DTNB, resulting in an increase in the optical density of the sample when read at an absorbance of 413 nm. To rule out non-specific compounds identified from the DTNB plate assay, we confirmed activity using a secondary thin layer chromatography (TLC) assay that directly measures the ability of LPAAT-β to catalyze the synthesis of PA from oleoyl-LPA and a fluorochrome-labeled (NBD) palmitoyl coenzyme A. Results: We screened a total of ten libraries containing 11204 compounds, and achieved a 1.2% hit rate for inhibition of >50% at the tested concentration (100μM for nine libraries; 10μM for one library). Further testing identified several compounds with IC50s ranging from 0.1-15 μM. One hit identified with an IC50 of 100 nM was chosen for further investigation and for synthesis of analogs to optimize potency and undertake SAR. We verified the activity of this hit and its analogs against LPAAT-β by determining their inhibitory activity of the synthesis of NBD-labeled PA in a TLC assay. Finally, we demonstrated that the best analog from this group inhibited the proliferation of several pancreatic cancer cell lines. Conclusions: We have developed a library of compounds which show inhibitory activity against LPAAT-β in a screening assay, and have confirmed their ability to inhibit LPAAT-β directly in a TLC assay which monitors the production of PA. These compounds show low micromolar activity against LPAAT-β in vitro, and micromolar activity against the proliferation of pancreatic cancer cell lines in culture. Citation Format: Michelle A. Blaskovich, Harsukh Gevariya, Nicholas J. Lawrence, Saïd Sebti, Gregory Springett. Identification and evaluation of small molecule inhibitors of lysophosphatidic acid acyltransferase-beta. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2013-2470

Computational evaluation of small molecule inhibitors of RGS4 to regulate the dopaminergic control of striatal LTD

Parkinson’s disease is a neurodegenerative disease which is the result of the degradation of the dopaminergic neurons in the substantia nigra pars compacta, leading to a disregulation of thalamocortical circuits. Traditional treatment involves the use of levodopa which increases the dopamine level in the striatum. There is a need for alternative non-dopamine therapy to prevent the side effects of the conventional drugs used. Recently small molecule inhibitors of RGS have become the prime candidates in studies related to regulating RGS by binding to its allosteric site and thus changing its structure. Through the docking studies we observed that these small molecule modulators of RGS4 make stable complexes with RGS4 when compared to native RGS4. The Gq(alpha)–RGS4–drug complexes are less stable. The increase in flexibility of the RGS4–drug complex could be the reason for the inability of the RGS4–drug complex to bind to the G protein. In our docking results, CCG63802 formed the most promising drug as a RGS4 inhibitor as it formed the most stable complex with RGS4 and also formed the least stable complex, Gq(alpha)–RGS4–CCG63802 complex. In our studies we evaluated the therapeutic potential of the small molecule inhibitors to provide a prospective treatment for Parkinson’s disease.

Pharmacophore-Based Virtual Screening and Biological Evaluation of Small Molecule Inhibitors for Protein Arginine Methylation

Protein arginine methyltransferases (PRMTs) are proved to play vital roles in chromatin remodeling, RNA metabolism, and signal transduction. Aberrant regulation of PRMT activity is associated with various pathological states such as cancer and cardiovascular disorders. Development and application of small molecule PRMT inhibitors will provide new avenues for therapeutic discovery. The combination of pharmacophore-based virtual screening methods with radioactive methylation assays provided six hits identified as inhibitors against the predominant arginine methyltransferase PRMT1 within micromolar potency. Two potent compounds, A9 and A36, exhibited the inhibitory effect by directly targeting substrate H4 other than PRMT1 and displayed even higher inhibition activity than the well-known PRMT inhibitors AMI-1. A9 significantly inhibits proliferation of castrate-resistant prostate cancer cells. Together, A9 may be a potential inhibitor against advanced hormone-independent cancers, and the work will provide clues for the future development of specific compounds that block the interaction of PRMTs with their targets.

Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid (Aβ) Self-Assembly

Dye-binding assays, such as those utilizing Congo red and thioflavin T, are among the most widely used tools to probe the aggregation of amyloidogenic biomolecules and for the evaluation of small molecule inhibitors of amyloid aggregation and fibrillization. A number of recent reports have indicated that these dye-binding assays could be prone to false positive effects when assessing inhibitors' potential toward Aβ peptides, species involved in Alzheimer's disease. Specifically, this review focuses on the application of thioflavin T for determining the efficiency of small molecule inhibitors of Aβ aggregation and addresses potential reasons that might be associated with the false positive effects in an effort to increase reliability of dye-binding assays.

Characterization of Pim Protein Kinases and Evaluation of Small Molecule Inhibitors in Multiple Myeloma

Abstract 2909Multiple myeloma (MM) is a plasma cell malignancy and is the second most common hematological neoplasm in the Western World. Despite the discovery of several highly effective chemotherapy agents, MM remains an incurable disease, suggesting the urgent need for better understanding the disease’s pathogenesis and for developing therapeutic agents that target novel molecular pathways. Pim (proviral insertion in murine lymphoma) kinases (Pim-1, -2 and -3) are constitutively active, oncogenic serine/threonine protein kinases that promote early transformation and tumor progression in hematological malignancies and in solid tumors including prostate cancer and colon cancer. In the current study, we characterized Pim expression and investigated the therapeutic potential of small molecule Pim kinase inhibitors in the treatment of MM. We found that Pim kinases are highly expressed in several MM cell lines (NCIH929, OPM-1, RPMI8226, U266 and MM1.R). Furthermore, Pim kinases were up-regulated in freshly isolated primary CD138+ myeloma cells (n = 4) when compared to CD138− cells (panel A). The predominant Pim kinase isoform (Pim-1, -2 or -3) varies among MM cell lines and among different patients. Two Pim kinase inhibitors, SMI-4a (selective small molecule Pim-1 and Pim-3 inhibitor) and 10058-F4 (Pim-3 and Pim-1 inhibitor) effectively inhibited myeloma cell growth, including steroid resistant MM1.R myeloma cells, with IC50s in the ∼10 mM range. Both SMI-4a and 10058-F4 induced apoptotic cell death in myeloma cells as measured by Annexin-V staining, caspase-9 activation and PARP cleavage in a dose dependent manner. To further dissect their mechanisms of action, we investigated the effects of SMI-4a and 10058-F4 on several cellular signaling pathways that are critical to the survival, cell cycle progression and proliferation of MM cells. Our data indicated that both SMI-4a and 10058-F4 reduce c-Myc protein expression levels and down-regulate the antiapoptotic Mcl-1 protein in MM cells (panel B). Our data also showed that MM cells treated with SMI-4a or 10058-F4 significantly reduced mTOR signaling as indicatd by decreased phosphorylation of 4E-BP1 and Cp70 S6 kinase α, the two common mTOR substrates. However, SMI-4a and 10058-F4 had no effect on the cell cycle regulation protein (p27Kip1) or heat shock protein 90. In summary, our data demonstrate that Pim inhibitors affect the survival and proliferation of myeloma cells and represent a potentially effective, novel strategy for the treatment of MM. We are currently performing in vivo studies to evaluate the therapeutic effects of Pim inhibitor in MM animal models (i.e, XPB-1s transgenic mouse model and NOD/SCID xenograft mouse model).This work was supported by MUSC Hollings Cancer Center Startup Fund, Hollings Cancer Center ACS IRG, and ASCO Conquer Cancer Foundation Career Development Award [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Design, synthesis and biological evaluation of small molecule inhibitors of CD4-gp120 binding based on virtual screening.

The low-molecular-weight compound JRC-II-191 inhibits infection of HIV-1 by blocking the binding of the HIV-1 envelope glycoprotein gp120 to the CD4 receptor and is therefore an important lead in the development of a potent viral entry inhibitor. Reported here is the use of two orthogonal screening methods, gold docking and ROCS shape-based similarity searching, to identify amine-building blocks that, when conjugated to the core scaffold, yield novel analogs that maintain similar affinity for gp120. Use of this computational approach to expand SAR produced analogs of equal inhibitory activity but with diverse capacity to enhance viral infection. The novel analogs provide additional lead scaffolds for the development of HIV-1 entry inhibitors that employ protein-ligand interactions in the vestibule of gp120 Phe 43 cavity.