Discovery of 3-(4-sulfamoylnaphthyl)pyrazolo[1,5-a]pyrimidines as potent and selective ALK2 inhibitors.

The pyrazolo[1,5-a]pyrimidine LDN-193189 is a potent inhibitor of activin receptor-like kinase 2 (ALK2) but is nonselective for highly homologous ALK3 and shows only modest kinome selectivity. Herein, we describe the discovery of a novel series of potent and selective ALK2 inhibitors by replacing the quinolinyl with a 4-(sulfamoyl)naphthyl, yielding ALK2 inhibitors that exhibit not only excellent discrimination versus ALK3 but also high kinome selectivity. In addition, the optimized compound 23 demonstrates good ADME and in vivo pharmacokinetic properties.

High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.

Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity.

Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 µM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 µM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50]=25 to 79 µM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

RUC-4: a novel αIIbß3 antagonist for prehospital therapy of myocardial infarction.

OBJECTIVE: Treatment of myocardial infarction within the first 1 to 2 hours with a thrombolytic agent, percutaneous coronary intervention, or an αIIbß3 antagonist decreases mortality and the later development of heart failure. We previously reported on a novel small molecule αIIbß3 antagonist, RUC-2, that has a unique mechanism of action. We have now developed a more potent and more soluble congener of RUC-2, RUC-4, designed to be easily administered intramuscularly by autoinjector to facilitate its use in the prehospital setting. Here, we report the properties of RUC-4 and the antiplatelet and antithrombotic effects of RUC-2 and RUC-4 in animal models. APPROACH AND RESULTS: RUC-4 was ≈ 20% more potent than RUC-2 in inhibiting human ADP-induced platelet aggregation and much more soluble in aqueous solutions (60-80 mg/mL). It shared RUC-2's specificity for αIIbß3 versus αVß3, did not prime the receptor to bind fibrinogen, or induce changes in ß3 identified by a conformation-specific monoclonal antibody. Both RUC-2 and RUC-4 prevented FeCl3-induced thrombotic occlusion of the carotid artery in mice and decreased microvascular thrombi in response to laser injury produced by human platelets infused into transgenic mice containing a mutated von Willebrand factor that reacts with human but not mouse platelets. Intramuscular injection of RUC-4 in nonhuman primates at 1.9 and 3.85 mg/kg led to complete inhibition of platelet aggregation within 15 minutes, with dose-dependent return of platelet aggregation after 4.5 to 24 hours. CONCLUSIONS: RUC-4 has favorable biochemical, pharmacokinetic, pharmacodynamic, antithrombotic, and solubility properties as a prehospital therapy of myocardial infarction, but the possibility of increased bleeding with therapeutic doses remains to be evaluated.

TCR signaling via Tec kinase ITK and interferon regulatory factor 4 (IRF4) regulates CD8+ T-cell differentiation.

CD8(+) T-cell development in the thymus generates a predominant population of conventional naive cells, along with minor populations of "innate" T cells that resemble memory cells. Recent studies analyzing a variety of KO or knock-in mice have indicated that impairments in the T-cell receptor (TCR) signaling pathway produce increased numbers of innate CD8(+) T cells, characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor, Eomesodermin (Eomes). One component of this altered development is a non-CD8(+) T cell-intrinsic role for IL-4. To determine whether reduced TCR signaling within the CD8(+) T cells might also contribute to this pathway, we investigated the role of the transcription factor, IFN regulatory factor 4 (IRF4). IRF4 is up-regulated following TCR stimulation in WT T cells; further, this up-regulation is impaired in T cells treated with a small-molecule inhibitor of the Tec family tyrosine kinase, IL-2 inducible T-cell kinase (ITK). In contrast to WT cells, activation of IRF4-deficient CD8(+) T cells leads to rapid and robust expression of Eomes, which is further enhanced by IL-4 stimulation. In addition, inhibition of ITK together with IL-4 increases Eomeso up-regulation. These data indicate that ITK signaling promotes IRF4 up-regulation following CD8(+) T-cell activation and that this signaling pathway normally suppresses Eomes expression, thereby regulating the differentiation pathway of CD8(+) T cells.

Multiple A2E treatments lead to melanization of rod outer segment-challenged ARPE-19 cells.

PURPOSE: Daily phagocytosis of outer segments (OSs) and retinoid recycling by the RPE lead to the accumulation of storage bodies in the RPE containing autofluorescent lipofuscin, which consists of lipids and bisretinoids such as A2E and its oxidation products. Accumulation of A2E and its oxidation products is implicated in the pathogenesis of several retinal degenerative diseases. However, A2E accumulates in the RPE during normal aging. In this study, we used a cell model to determine the homeostatic mechanisms of RPE cells in response to A2E accumulation. METHODS: To distinguish between pathologic and normal responses of the RPE to A2E accumulation, we treated established ARPE-19 cells (cultured for 3 weeks after reaching confluence) with low micromolar amounts of A2E for several weeks. We compared the lysosomal function, lysosomal pH, degree of OS digestion, and melanization of the treated cells to untreated control cells in response to a challenge of purified rod OSs (ROSs). A2E was analyzed with high-performance liquid chromatography (HPLC); and A2E and melanin were identified with mass spectrometry. RESULTS: We found that post-confluent ARPE-19 cells took up and accumulated A2E under dim light conditions. Spectral analysis of the HPLC separations and mass spectrometry showed that A2E-fed cells contained A2E and oxidized A2E (furan-A2E). A2E accumulation led to a modest increase (up to 0.25 unit) in lysosomal pH in these cells. The specific activity of cathepsin D and lysosomal acid phosphatase was reduced in the A2E-treated cells, but ROS degradation was not impaired. We found that, upon challenge with ROSs, melanin pigment was induced in the lysosomal fraction of the A2E-treated ARPE-19 cells. Thus, the ARPE-19 cells responded to the A2E treatment and ROS challenge by producing a melanin-containing lysosome fraction. We speculate that this prevents them from becoming impaired in OS processing. CONCLUSIONS: We used a modified ARPE-19 cell model in which melanization was elicited as a response to chronic accumulation of A2E. We found that although A2E treatment led, as has been previously reported, to modest lysosomal alkalinization and lysosomal impairment of ARPE-19 cells, a potential homeostatic mechanism may involve production of a special type of lysosomes containing melanin.

Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis.

Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

A novel class of ion displacement ligands as antagonists of the αIIbß3 receptor that limit conformational reorganization of the receptor.

A collection of αIIbß3 integrin receptor antagonists possessing a unique MIDAS metal ion displacement mechanism of action is presented. Insight into these agents' structure-activity relationships, binding modality, and pharmacokinetic and pharmacodynamic profiles highlight the potential of these small molecule ion displacement ligands as attractive candidates for clinical development.

CP-690,550, a therapeutic agent, inhibits cytokine-mediated Jak3 activation and proliferation of T cells from patients with ATL and HAM/TSP.

The retrovirus, human T-cell-lymphotrophic virus-1 (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-I-encoded protein tax constitutively activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems that in turn activate the Jak3 (Janus kinase 3)/STAT5 (signal transducers and activators of transcription 5) pathway, suggesting a therapeutic strategy that involves targeting Jak3. We evaluated the action of the Jak3 inhibitor CP-690,550 on cytokine dependent ex vivo proliferation that is characteristic of peripheral blood mononuclear cells (PBMCs) from select patients with smoldering or chronic subtypes of ATL, or from those with HAM/TSP whose PBMCs are associated with autocrine/paracrine pathways that involve the production of IL-2, IL-9, IL-15, and their receptors. CP-690,550 at 50 nM inhibited the 6-day ex vivo spontaneous proliferation of PBMCs from ATL and HAM/TSP patients by 67.1% and 86.4%, respectively. Furthermore, CP-690,550 inhibited STAT5 phosphorylation in isolated ATL T cells ex vivo. Finally, in an in vivo test of biological activity, CP-690,550 treatment of mice with a CD8 T-cell IL-15-transgenic leukemia that manifests an autocrine IL-15/IL-15Rα pathway prolonged the survival duration of these tumor-bearing mice. These studies support further evaluation of the Jak3 inhibitor CP-690,550 in the treatment of select patients with HTLV-I-associated ATL and HAM/TSP.

Calcium-sensing receptor is a physiologic multimodal chemosensor regulating gastric G-cell growth and gastrin secretion.

The calcium-sensing receptor (CaR) is the major sensor and regulator of extracellular Ca(2+), whose activity is allosterically regulated by amino acids and pH. Recently, CaR has been identified in the stomach and intestinal tract, where it has been proposed to function in a non-Ca(2+) homeostatic capacity. Luminal nutrients, such as Ca(2+) and amino acids, have been recognized for decades as potent stimulants for gastrin and acid secretion, although the molecular basis for their recognition remains unknown. The expression of CaR on gastrin-secreting G cells in the stomach and their shared activation by Ca(2+), amino acids, and elevated pH suggest that CaR may function as the elusive physiologic sensor regulating gastrin and acid secretion. The genetic and pharmacologic studies presented here comparing CaR-null mice and wild-type littermates support this hypothesis. Gavage of Ca(2+), peptone, phenylalanine, Hepes buffer (pH 7.4), and CaR-specific calcimimetic, cinacalcet, stimulated gastrin and acid secretion, whereas the calcilytic, NPS 2143, inhibited secretion only in the wild-type mouse. Consistent with known growth and developmental functions of CaR, G-cell number was progressively reduced between 30 and 90 d of age by more than 65% in CaR-null mice. These studies of nutrient-regulated G-cell gastrin secretion and growth provide definitive evidence that CaR functions as a physiologically relevant multimodal sensor. Medicinals targeting diseases of Ca(2+) homeostasis should be reviewed for effects outside traditional Ca(2+)-regulating tissues in view of the broader distribution and function of CaR.

Selective targeting of ITK blocks multiple steps of HIV replication.

Treatment for HIV has relied on the use of antiretroviral agents that can be subject to the development of resistant viruses. The study of inhibitors directed against cellular proteins required for HIV replication is therefore of growing interest. Inducible T cell kinase (ITK) is a Tec family tyrosine kinase that regulates T cell receptor (TCR)-induced activation of PLCgamma-1, Ca(2+) mobilization and transcription factor activation, and actin rearrangement downstream of both TCR and chemokine receptors. Because productive infection of T cells with HIV requires T cell activation, chemokine receptors and actin reorganization, we asked whether ITK affects HIV infection using ITK-specific siRNA, a kinase-inactive ITK mutant or an ITK inhibitor. We demonstrate that loss of ITK function resulted in marked reductions in intracellular p24 levels upon HIV infection. Loss of ITK function after establishment of HIV infection also decreased virus spread within the culture. Inhibition of ITK did not affect expression of the HIV coreceptors CD4 or CXCR4 but partially blocked HIV viral entry, an effect that correlated with decreased actin polarization to gp120. Additionally, ITK was required for efficient HIV transcription, and overexpression of ITK increased both viral transcription and virus-like particle formation. Our data suggest that inhibition of ITK blocks HIV infection by affecting multiple steps of HIV replication.

Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as activators of the tumor cell specific M2 isoform of pyruvate kinase.

Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure-activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.

Targeting AML-associated FLT3 mutations with a type I kinase inhibitor.

Tyrosine kinase domain (TKD) mutations contribute to acquired resistance to FMS-like tyrosine kinase 3 (FLT3) inhibitors used to treat FLT3-mutant acute myeloid leukemia (AML). We report a cocrystal structure of FLT3 with a type I inhibitor, NCGC1481, that retained potent binding and activity against FLT3 TKD and gatekeeper mutations. Relative to the current generation of advanced FLT3 inhibitors, NCGC1481 exhibited superior antileukemic activity against the common, clinically relevant FLT3-mutant AML cells in vitro and in vivo.

Application of in vitro Drug Metabolism Studies in Chemical Structure Optimization for the Treatment of Fibrodysplasia Ossificans Progressiva (FOP).

Currently no approved treatment exists for fibrodysplasia ossificans progressiva (FOP) patients, and disease progression results in severe restriction of joint function and premature mortality. LDN-193189 has been demonstrated to be efficacious in a mouse FOP disease model after oral administration. To support species selection for drug safety evaluation and to guide structure optimization for back-up compounds, in vitro metabolism of LDN-193189 was investigated in liver microsome and cytosol fractions of mouse, rat, dog, rabbit, monkey and human. Metabolism studies included analysis of reactive intermediate formation using glutathione and potassium cyanide (KCN) and analysis of non-P450 mediated metabolites in cytosol fractions of various species. Metabolite profiles and metabolic soft spots of LDN-193189 were elucidated using LC/UV and mass spectral techniques. The in vitro metabolism of LDN-193189 was significantly dependent on aldehyde oxidase, with formation of the major NIH-Q55 metabolite. The piperazinyl moiety of LDN-193189 was liable to NADPH-dependent metabolism which generated reactive iminium intermediates, as confirmed through KCN trapping experiments, and aniline metabolites (M337 and M380), which brought up potential drug safety concerns. Subsequently, strategies were employed to avoid metabolic liabilities leading to the synthesis of Compounds 1, 2, and 3. This study demonstrated the importance of metabolite identification for the discovery of novel and safe drug candidates for the treatment of FOP and helped medicinal chemists steer away from potential metabolic liabilities.

Target Deconvolution of a Multikinase Inhibitor with Antimetastatic Properties Identifies TAOK3 as a Key Contributor to a Cancer Stem Cell-Like Phenotype.

Pancreatic cancer remains an incurable condition. Its progression is driven, in part, by subsets of cancer cells that evade the cytotoxic effects of conventional chemotherapies. These cells are often low-cycling, multidrug resistant, and adopt a stem cell-like phenotype consistent with the concept of cancer stem cells (CSC). To identify drugs impacting on tumor-promoting CSCs, we performed a differential high-throughput drug screen in pancreatic cancer cells cultured in traditional (2D) monolayers versus three-dimensional (3D) spheroids which replicate key elements of the CSC model. Among the agents capable of killing cells cultured in both formats was a 1H-benzo[d]imidazol-2-amine-based inhibitor of IL2-inducible T-cell kinase (ITK; NCGC00188382, inhibitor #1) that effectively mediated growth inhibition and induction of apoptosis in vitro, and suppressed cancer progression and metastasis formation in vivo An examination of this agent's polypharmacology via in vitro and in situ phosphoproteomic profiling demonstrated an activity profile enriched for mediators involved in DNA damage repair. Included was a strong inhibitory potential versus the thousand-and-one amino acid kinase 3 (TAOK3), CDK7, and aurora B kinases. We found that cells grown under CSC-enriching spheroid conditions are selectively dependent on TAOK3 signaling. Loss of TAOK3 decreases colony formation, expression of stem cell markers, and sensitizes spheroids to the genotoxic effect of gemcitabine, whereas overexpression of TAOK3 increases stem cell traits including tumor initiation and metastasis formation. By inactivating multiple components of the cell-cycle machinery in concert with the downregulation of key CSC signatures, inhibitor #1 defines a distinctive strategy for targeting pancreatic cancer cell populations.

Overcoming adaptive therapy resistance in AML by targeting immune response pathways.

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course; however, most patients will relapse because of target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. Here, we describe mechanisms of adaptive resistance in FMS-like receptor tyrosine kinase (FLT3)-mutant acute myeloid leukemia (AML) by examining integrative in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). We identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i and showed that innate immune pathway activation via the interleukin-1 receptor-associated kinase 1 and 4 (IRAK1/4) complex contributes to adaptive resistance in FLT3-mutant AML cells. To overcome this adaptive resistance mechanism, we developed a small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The multikinase FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cells in vitro and in vivo and displayed superior efficacy as compared to current targeted FLT3 therapies. These findings uncover a polypharmacologic strategy for overcoming adaptive resistance to therapy in AML by targeting immune stress response pathways.

A low-molecular-weight antagonist for the human thyrotropin receptor with therapeutic potential for hyperthyroidism.

Low-molecular-weight (LMW) antagonists for TSH receptor (TSHR) may have therapeutic potential as orally active drugs to block stimulating antibodies (TsAbs) in Graves' hyperthyroidism. We describe an approach to identify LMW ligands for TSHR based on Org41841, a LMW partial agonist for the LH/choriogonadotropin receptor and TSHR. We used molecular modeling and functional experiments to guide the chemical modification of Org41841. We identified an antagonist (NIDDK/CEB-52) that selectively inhibits activation of TSHR by both TSH and TsAbs. Whereas initially characterized in cultured cells overexpressing TSHRs, the antagonist was also active under more physiologically relevant conditions in primary cultures of human thyrocytes expressing endogenous TSHRs in which it inhibited TSH- and TsAb-induced up-regulation of mRNA transcripts for thyroperoxidase. Our results establish this LMW compound as a lead for the development of higher potency antagonists and serve as proof of principle that LMW ligands that target TSHR could serve as drugs in patients with Graves' disease.

Evaluation of small-molecule modulators of the luteinizing hormone/choriogonadotropin and thyroid stimulating hormone receptors: structure-activity relationships and selective binding patterns.

The substituted thieno[2,3-d]pyrimidine 3 (Org 41841), a partial agonist for the luteinizing hormone/choriogonadotropin receptor (LHCGR) and the closely related thyroid-stimulating hormone receptor (TSHR), was fundamentally altered, and the resulting analogues were analyzed for their potencies, efficacies, and specificities at LHCGR and TSHR. Chemical modification of the parent compound combined with prior mutagenesis of TSHR provided compelling experimental evidence in support of computational models of 3 binding to TSHR and LHCGR within their transmembrane cores. Biochemical analysis of a specific modification to the chemical structure of 3 provides additional evidence of a H-bond between the ligand and a glutamate residue in transmembrane helix 3, which is conserved in both receptors. Several key interactions were surveyed to determine their respective biochemical roles in terms of both van der Waals dimensions and hydrogen bond capacity and the respective relationship to biological activity.