Cytochrome P450 3A4 is an important mediator of drug catabolism that can be regulated by the steroid and xenobiotic receptor (SXR). We show here that SXR also regulates drug efflux by activating expression of the gene MDR1, which encodes the protein P-glycoprotein (ABCB1). Paclitaxel (Taxol), a commonly used chemotherapeutic agent, activated SXR and enhanced P-glycoprotein-mediated drug clearance. In contrast, docetaxel (Taxotere), a closely related antineoplastic agent, did not activate SXR and displayed superior pharmacokinetic properties. Docetaxel's silent properties reflect its inability to displace transcriptional corepressors from SXR. We also found that ET-743, a potent antineoplastic agent, suppressed MDR1 transcription by acting as an inhibitor of SXR. These findings demonstrate how the molecular activities of SXR can be manipulated to control drug clearance.

It has recently been shown that the neurological mutant mouse staggerer (sg) harbors a deletion within the Rora gene that encodes the orphan nuclear receptor ROR alpha. This deletion removes an exon encoding part of the ligand binding domain of the putative receptor, generating an ROR alpha truncated protein (ROR alpha(sg)). It is unknown whether sg acts as a null or highly hypomorphic allele. To address this question, we have generated a null mutation of Rora by targeted disruption of its DNA binding domain in ES cells. The Rora-/- mice are viable but display tremor, body imbalance, small size and die between 3-4 weeks, similar to the sg mouse. Histological examination of the cerebellum of Rora-/- and sg mice showed similar defects, including small size and fewer ectopically localized Purkinje cells. Northern blot analysis of cerebellar RNA showed that ROR alpha transcripts are still expressed in the Rora-/- and sg mutants, although with altered mobilities. However, the cerebellum of the Rora-/- mutant does not express the ROR alpha protein. Attempts to complement the defect of the Rora-/- with sg failed, demonstrating conclusively that the sg defects are caused by the absence of functional ROR alpha.

Unlike classical nuclear receptors that require ligand for transcriptional activity, the constitutive androstane receptor (CAR) is active in the absence of ligand. To determine the molecular contacts that underlie this constitutive activity, we created a three-dimensional model of CAR and verified critical structural features by mutational analysis. We found that the same motifs that facilitate ligand-dependent activity in classical receptors also mediated constitutive activity in CAR. This raises a critical question: how are these motifs maintained in an active conformation in unliganded CAR? The model identified several novel interactions that account for this activity. First, CAR possesses a short loop between helix 11 and the transactivation domain (helix 12), as well as a short carboxy-terminal helix. Together, these features favor ligand-independent docking of the transactivation domain in a position that is characteristic of ligand-activated receptors. Second, this active conformation is further stabilized by a charge-charge interaction that anchors the carboxy-terminal activation domain to helix 4. Mutational analysis of these interactions provides direct experimental support for this model. We also show that ligand-mediated repression of constitutive activity reflects both a displacement of coactivator and a recruitment of corepressor. Our data demonstrate that CAR utilizes the same conserved structural motifs and coregulator proteins as originally defined for classical nuclear receptors. Despite these remarkable similarities, our model demonstrates how a few critical changes in CAR can dramatically reverse the transcriptional activity of this protein.Nuclear hormone receptors are transcription factors essential for virtually all aspects of physiology, including normal differentiation, development, and homeostasis. The transcriptional activity of these receptors is modulated by small lipophilic ligands, including the classical steroid hormones, thyroid hormone, retinoids, and other lipid metabolites (33). Upon binding ligand, classical nuclear receptors undergo a conformation change that results in the recruitment or displacement of a variety of coregulator proteins (15, 55). These coregulators include coactivators (PBP/DRIP205/TRAP220/ TRIP2, SRC-1/NCoA-1, GRIP1/TIF2/NCoA-2, and ACTR/ pCIP/AIB1/NCoA-3) (2,4,19,30,32,38,47,56,59) and corepressors (nuclear receptor corepressor [NCoR] and silencing mediator of retinoic acid and thyroid hormone receptors [SMRT]) (5, 21, 42). Both classes of coregulators utilize ␣-helical motifs (receptor interaction domains [RIDs]) to make direct contacts with a hydrophobic cleft (8, 11, 37, 43) on the surface of nuclear receptors (18,22,47). These coregulators form complexes with other proteins that function either by remodeling chromatin or by providing a bridge between the nuclear receptor and the basal transcription machinery. These interactions allow nuclear receptor ligands to activate or repress transcription of specific target genes (17).Nuclear receptors have a c...

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division.

c-Met is a receptor tyrosine kinase often deregulated in human cancers, thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met, whereas other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers.

The orphan nuclear receptor SXR coordinately regulates drug clearance in response to a wide variety of xenobiotic compounds. This signaling system protects the body from exposure to toxic compounds; however, it can also pose a severe barrier to drug therapy. We now demonstrate that the human immunodeficiency virus (HIV) protease inhibitor ritonavir binds SXR and activates its target genes. This represents an example of a commonly used therapeutic agent that effectively activates SXR. We also show that other protease inhibitors are weaker (saquinavir) or unable to activate SXR (nelfinavir, indinavir) thus defining analogs that fail to induce SXR-regulated clearance pathways. Interestingly, HIV protease inhibitors are distinct from previously known SXR ligands in that they are peptide mimetic compounds. This expands the ligand specificity of SXR to include this unique chemical class whose pharmaceutical significance is expanding. Finally, we show that SXR ligands activate expression of multiple resistance protein 2, a critical regulator of bile flow and biliary drug excretion. These findings have important implications for the role of SXR in regulating drug clearance and hepatic disorders associated with impaired bile flow.

Introduction: The safety and efficacy of bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor b (TGF-b) receptor II (a TGF-b "trap") fused to a human immunoglobulin G1 antibody blocking programmed deathligand 1 (PD-L1), was evaluated in patients with advanced NSCLC.Methods: This expansion cohort of NCT02517398, an ongoing, phase 1, open-label trial, includes 80 patients with advanced NSCLC that progressed after platinum doublet therapy or after platinum-based adjuvant or neoadjuvant treatment and those who also have not received previous immunotherapy. Patients were randomized at a one-to-one ratio to receive either bintrafusp alfa 500 mg or the recommended phase 2 dosage of 1200 mg every 2 weeks. The primary end point was the best overall response (by Response Evaluation Criteria in Solid Tumors 1.1 as adjudicated by independent review committee) and was assessed by the objective response rate (ORR).Results: A total of 80 patients were randomized to receive bintrafusp alfa 500 or 1200 mg (n ¼ 40 each). Median follow-up was 51.9 weeks (IQR, 19.6-74.0). The ORR in all patients was 21.3% (17 of 80). The ORR was 17.5% (seven of 40) and 25.0% (10 of 40) for the 500 mg dose and the 1200 mg dose (recommended phase 2 dose), respectively. At the 1200 mg dose, patients with PD-L1-positive and PD-L1-high (80% expression on tumor cells) had ORRs of 36.0% (10 of 27) and 85.7% (six of seven), respectively. Treatment-related adverse events occurred in 55 of the 80 patients (69%) and were graded as greater than or equal to 3 in 23 of the 80 patients (29%). Of the 80 patients, eight (10%) had a treatment-related adverse event that led to treatment discontinuation; no treatment-related deaths occurred.Conclusions: Bintrafusp alfa had encouraging efficacy and manageable tolerability in patients with NSCLC previously treated with platinum.

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors. Cancer Res; 71(17); 5818-26. Ó2011 AACR.