An anti-CTLA-4 heavy chain-only antibody with enhanced Treg depletion shows excellent preclinical efficacy and safety profile.

The value of anti-CTLA-4 antibodies in cancer therapy is well established. However, the broad application of currently available anti-CTLA-4 therapeutic antibodies is hampered by their narrow therapeutic index. It is therefore challenging and attractive to develop the next generation of anti-CTLA-4 therapeutics with improved safety and efficacy. To this end, we generated fully human heavy chain-only antibodies (HCAbs) against CTLA-4. The hIgG1 Fc domain of the top candidate, HCAb 4003-1, was further engineered to enhance its regulatory T (Treg) cell depletion effect and to decrease its half-life, resulting in HCAb 4003-2. We tested these HCAbs in in vitro and in vivo experiments in comparison with ipilimumab and other anti-CTLA4 antibodies. The results show that human HCAb 4003-2 binds human CTLA-4 with high affinity and potently blocks the binding of B7-1 (CD80) and B7-2 (CD86) to CTLA-4. The results also show efficient tumor penetration. HCAb 4003-2 exhibits enhanced antibody-dependent cellular cytotoxicity function, lower serum exposure, and more potent anti-tumor activity than ipilimumab in murine tumor models, which is partly driven by a substantial depletion of intratumoral Tregs. Importantly, the enhanced efficacy combined with the shorter serum half-life and less systemic drug exposure in vivo potentially provides an improved therapeutic window in cynomolgus monkeys and preliminary clinical applications. With its augmented efficacy via Treg depletion and improved safety profile, HCAb 4003-2 is a promising candidate for the development of next generation anti-CTLA-4 therapy.

Design and synthesis of orally bioavailable aminopyrrolidinone histone deacetylase 6 inhibitors.

Histone deacetylase 6 (HDAC6) removes the acetyl group from lysine residues in a number of non-histone substrates and plays important roles in microtubule dynamics and chaperone activities. There is growing interest in identifying HDAC6-selective inhibitors as chemical biology tools and ultimately as new therapeutic agents. Herein we report the design, synthesis, and phenotypic screening of a novel class of 3-aminopyrrolidinone-based hydroxamic acids as HDAC6 inhibitors. In particular, the α-methyl-substituted enantiomer 33 (3-S) showed significant in-cell tubulin acetylation (Tub-Ac) with an EC50 of 0.30 µM but limited impact on p21 levels at various concentrations. In enzyme inhibition assays, 33 demonstrated high selectivity for HDAC6 with an IC50 of 0.017 µM and selectivity indexes of 10 against HDAC8 and over 4000 against HDAC1-3 isoforms. Moreover, 33 has suitable drug metabolism and pharmacokinetics properties compared with other hydroxamic acid-based HDAC inhibitors, warranting further biological studies and development as a selective HDAC6 inhibitor.

Targeting Hippo pathway by specific interruption of YAP-TEAD interaction using cyclic YAP-like peptides.

Hippo signaling pathway is emerging as a novel target for anticancer therapy because it plays key roles in organ size control and tumorigenesis. As the downstream effectors, Yes-associated protein (YAP)-transcriptional enhancer activation domain family member (TEAD) association is essential for YAP-driven oncogenic activity, while TEAD is largely dispensable for normal tissue growth. We present the design of YAP-like peptides (17mer) to occupy the interface 3 on TEAD. Introducing cysteines at YAP sites 87 and 96 can induce disulfide formation, as confirmed by crystallography. The engineered peptide significantly improves the potency in disrupting YAP-TEAD interaction in vitro. To confirm that blocking YAP-TEAD complex formation by directly targeting on TEAD is a valid approach, we report a significant reduction in tumor growth rate in a hepatocellular carcinoma xenograft model after introducing the dominant-negative mutation (Y406H) of TEAD1 to abolish YAP-TEAD interaction. Our results suggest that targeting TEAD is a promising strategy against YAP-induced oncogenesis.

Structure-Based Design and Synthesis of Potent Cyclic Peptides Inhibiting the YAP-TEAD Protein-Protein Interaction.

The YAP-TEAD protein-protein interaction (PPI) mediates the oncogenic function of YAP, and inhibitors of this PPI have potential usage in treatment of YAP-involved cancers. Here we report the design and synthesis of potent cyclic peptide inhibitors of the YAP-TEAD interaction. A truncation study of YAP interface 3 peptide identified YAP(84-100) as a weak peptide inhibitor (IC50 = 37 µM), and an alanine scan revealed a beneficial mutation, D94A. Subsequent replacement of a native cation-π interaction with an optimized disulfide bridge for conformational constraint and synergistic effect between macrocyclization and modification at positions 91 and 93 greatly boosted inhibitory activity. Peptide 17 was identified with an IC50 of 25 nM, and the binding affinity (K d = 15 nM) of this 17mer peptide to TEAD1 proved to be stronger than YAP(50-171) (K d = 40 nM).

Identification of a novel aminotetralin class of HDAC6 and HDAC8 selective inhibitors.

Herein we report the identification of a novel class of HDAC6 and HDAC8 selective inhibitors through a unique chemistry and phenotypic screening strategy. Tetrahydroisoquinoline 12 was identified as a potent HDAC6 and HDAC8 dual inhibitor from a focused library through cellular tubulin acetylation and p21 induction screening assays. Scaffold hopping from 12 led to the discovery of an aminotetralin class of HDAC inhibitors. In particular, the 3-R stereoisomer 32 showed highly potent inhibition against HDAC6 and HDAC8 with IC50 values of 50 and 80 nM, respectively. Treatment of neuroblastoma BE(2)C cells with 32 resulted in elevated levels of acetylated tubulin, TrkA, and neurite outgrowth with only marginal effects on p21 induction and histone H3 acetylation. Consistent with its weak enzymatic inhibition of HDAC1, 32 showed significantly less cytotoxicity than SAHA and moderately inhibited the growth of myeloma NCI-H929 and OPM-2 cells.

Pharmacokinetic optimization of class-selective histone deacetylase inhibitors and identification of associated candidate predictive biomarkers of hepatocellular carcinoma tumor response.

Herein, we describe the pharmacokinetic optimization of a series of class-selective histone deacetylase (HDAC) inhibitors and the subsequent identification of candidate predictive biomarkers of hepatocellular carcinoma (HCC) tumor response for our clinical lead using patient-derived HCC tumor xenograft models. Through a combination of conformational constraint and scaffold hopping, we lowered the in vivo clearance (CL) and significantly improved the bioavailability (F) and exposure (AUC) of our HDAC inhibitors while maintaining selectivity toward the class I HDAC family with particular potency against HDAC1, resulting in clinical lead 5 (HDAC1 IC50 = 60 nM, mouse CL = 39 mL/min/kg, mouse F = 100%, mouse AUC after single oral dose at 10 mg/kg = 6316 h·ng/mL). We then evaluated 5 in a biomarker discovery pilot study using patient-derived tumor xenograft models, wherein two out of the three models responded to treatment. By comparing tumor response status to compound tumor exposure, induction of acetylated histone H3, candidate gene expression changes, and promoter DNA methylation status from all three models at various time points, we identified preliminary candidate response prediction biomarkers that warrant further validation in a larger cohort of patient-derived tumor models and through confirmatory functional studies.

Induction of Ca²+-driven apoptosis in chronic lymphocytic leukemia cells by peptide-mediated disruption of Bcl-2-IP3 receptor interaction.

Bcl-2 contributes to the pathophysiology and therapeutic resistance of chronic lymphocytic leukemia (CLL). Therefore, developing inhibitors of this protein based on a thorough understanding of its mechanism of action is an active and promising area of inquiry. One approach centers on agents (eg, ABT-737) that compete with proapoptotic members of the Bcl-2 protein family for binding in the hydrophobic groove formed by the BH1-BH3 domains of Bcl-2. Another region of Bcl-2, the BH4 domain, also contributes to the antiapoptotic activity of Bcl-2 by binding to the inositol 1,4,5-trisphosphate receptor (IP3R) Ca²(+) channel, inhibiting IP(3)-dependent Ca²(+) release from the endoplasmic reticulum. We report that a novel synthetic peptide, modeled after the Bcl-2-interacting site on the IP3R, binds to the BH4 domain of Bcl-2 and functions as a competitive inhibitor of the Bcl-2-IP3R interaction. By disrupting the Bcl-2-IP3R interaction, this peptide induces an IP3R-dependent Ca²(+) elevation in lymphoma and leukemia cell lines and in primary CLL cells. The Ca²(+) elevation evoked by this peptide induces apoptosis in CLL cells, but not in normal peripheral blood lymphocytes, suggesting the involvement of the Bcl-2-IP3R interaction in the molecular mechanism of CLL and indicating the potential merit of targeting this interaction therapeutically.

Glucocorticoid-mediated inhibition of Lck modulates the pattern of T cell receptor-induced calcium signals by down-regulating inositol 1,4,5-trisphosphate receptors.

Glucocorticoids are potent immunosuppressive agents that block upstream signaling events required for T cell receptor (TCR) activation. However, the mechanism by which glucocorticoids inhibit downstream responses, such as inositol 1,4,5-trisphosphate (IP(3))-induced calcium signals, is not completely understood. Here we demonstrate that low concentrations of dexamethasone rapidly convert transient calcium elevations to oscillations after strong TCR stimulation. Dexamethasone converted the pattern of calcium signaling by inhibiting the Src family kinase Lck, which was shown to interact with and positively regulate Type I IP(3) receptor. In addition, low concentrations of dexamethasone were sufficient to inhibit calcium oscillations and interleukin-2 mRNA after weak TCR stimulation. Together, these findings indicate that by inhibiting Lck and subsequently down-regulating IP(3) receptors, glucocorticoids suppress immune responses by weakening the strength of the TCR signal.

The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP3 receptor.

Although the presence of a BH4 domain distinguishes the antiapoptotic protein Bcl-2 from its proapoptotic relatives, little is known about its function. BH4 deletion converts Bcl-2 into a proapoptotic protein, whereas a TAT-BH4 fusion peptide inhibits apoptosis and improves survival in models of disease due to accelerated apoptosis. Thus, the BH4 domain has antiapoptotic activity independent of full-length Bcl-2. Here we report that the BH4 domain mediates interaction of Bcl-2 with the inositol 1,4,5-trisphosphate (IP3) receptor, an IP3-gated Ca(2+) channel on the endoplasmic reticulum (ER). BH4 peptide binds to the regulatory and coupling domain of the IP3 receptor and inhibits IP3-dependent channel opening, Ca(2+) release from the ER, and Ca(2+)-mediated apoptosis. A peptide inhibitor of Bcl-2-IP3 receptor interaction prevents these BH4-mediated effects. By inhibiting proapoptotic Ca(2+) signals at their point of origin, the Bcl-2 BH4 domain has the facility to block diverse pathways through which Ca(2+) induces apoptosis.

Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol 1,4,5-trisphosphate receptor.

Bcl-2 is the founding member of a large family of apoptosis regulating proteins. Bcl-2 is a prime target for novel therapeutics because it is elevated in many forms of cancer and contributes to cancer progression and therapy resistance based on its ability to inhibit apoptosis. Bcl-2 interacts with proapoptotic members of the Bcl-2 family to inhibit apoptosis and small molecules that disrupt this interaction have already entered the cancer therapy arena. A separate function of Bcl-2 is to inhibit Ca2+ signals that promote apoptosis. This function is mediated through interaction of the Bcl-2 BH4 domain with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel. A novel peptide inhibitor of this interaction enhances proapoptotic Ca2+ signals. In preliminary experiments this peptide enhanced ABT-737 induced apoptosis in chronic lymphocytic leukemia cells. These findings draw attention to the BH4 domain as a potential therapeutic target. This review summarizes what is currently known about the BH4 domain of Bcl-2, its interaction with the IP3R and other proteins, and the part it plays in Bcl-2's anti-apoptotic function. In addition, we speculate on how the BH4 domain of Bcl-2 can be targeted therapeutically not only for diseases associated with apoptosis resistance, but also for diseases associated with accelerated cell death.

Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals.

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

Apoptosis inhibition by Bcl-2 gives way to autophagy in glucocorticoid-treated lymphocytes.

Glucocorticosteroid hormones, including prednisone and dexamethasone (Dex), have been used to treat lymphoid malignancies for many years because they readily induce apoptosis in immature lymphocytes lacking Bcl-2. However, elevated expression of the anti-apoptotic protein Bcl-2 inhibits apoptosis and contributes to glucocorticoid resistance. Using the Bcl-2-negative WEHI7.2 lymphoma line as an experimental model, we found that Dex not only induces apoptosis but also induces autophagy. The caspase inhibitor Z-VAD-fmk inhibited apoptosis but not autophagy in Dex-treated cells. Bcl-2 overexpression inhibited Dex-induced apoptosis even more potently than Z-VAD-fmk and, contrary to previous reports, Bcl-2 neither interacted with Beclin-1 nor inhibited autophagy. Rather, Bcl-2 overexpression facilitated detection of Dex-induced autophagy by both steady state methods and flux measurements, ostensibly due to apoptosis inhibition. Autophagy contributed to prolonged survival of Bcl-2-positive lymphoma cells following Dex treatment, as survival was reduced when autophagy was inhibited by 3-methyladenine. These findings emphasize the important interplay between apoptosis and autophagy and suggest a novel mechanism by which Bcl-2, which is frequently elevated in lymphoid malignancies, contributes to glucocorticoid resistance and survival of lymphoma cells.

Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis.

Bcl-2 family members are important regulators of cell survival and cell death. Researchers have focused mainly on mitochondria, where both proapoptotic and antiapoptotic family members function to regulate the release of cytochrome c and other mediators of apoptosis. However, as reviewed here, Bcl-2 family members also operate on another front, the endoplasmic reticulum (ER), to both positively and negatively regulate the release of Ca2+. There is abundant evidence that Ca2+ signals trigger apoptosis in response to a wide variety of agents and conditions. Conversely, Ca2+ signals can also mediate cell survival. Recent findings indicate that Bcl-2 interacts with inositol 1,4,5-trisphosphate (IP3) receptor Ca2+ channels on the ER, regulating their opening in response to IP3- and thus inhibiting IP3-mediated Ca2+ signals that induce apoptosis while enhancing Ca2+ signals that support cell survival.

Isolation and characterization of a novel human thioredoxin-like gene hTLP19 encoding a secretory protein.

A novel human gene, named as hTLP19, was isolated and characterized as secretory protein by combining bioinformatics tools with experiments. The hTLP19 encodes 172 amino acid residues with signal peptide in its N-terminal and a thioredoxin (Trx) domain that is homologous with some genes in Mus musculus, Xenopus laevis, etc. Moreover, the result from insulin reduction assay indicated that the hTLP19 protein has Trx enzymatic activity. By comparing full-length cDNA with human genomic DNA, the hTLP19 gene might have seven coding exons spanning 35 kb of genomic DNA on the human chromosome 1p32.3. Northern blot analysis showed that human hTLP19 is expressed widely in many tissues with 1.6 kb band and extra 1.2 kb band in placenta. Subcellular localization and immunoblotting assays indicated that hTLP19 might be secreted out of cell through trans-Golgi network (TGN).

Evolutionary and biomedical implications of a Schistosoma japonicum complementary DNA resource.

Schistosoma japonicum causes schistosomiasis in humans and livestock in the Asia-Pacific region. Knowledge of the genome of this parasite should improve understanding of schistosome-host interactions, biomedical aspects of schistosomiasis and invertebrate evolution. We assigned 43,707 expressed sequence tags (ESTs) derived from adult S. japonicum and their eggs to 13,131 gene clusters. Of these, 35% shared no similarity with known genes and 75% had not been reported previously in schistosomes. Notably, S. japonicum encoded mammalian-like receptors for insulin, progesterone, cytokines and neuropeptides, suggesting that host hormones, or endogenous parasite homologs, could orchestrate schistosome development and maturation and that schistosomes modulate anti-parasite immune responses through inhibitors, molecular mimicry and other evasion strategies.

Cloning and characterization of a novel human secretory protein: secretogranin III.

We have cloned a new member of the granin family, termed human secretogranin III (SgIII), that encodes 468 amino acid residues. The human SgIII protein possesses an N-terminal signal peptide, seven dibasic sites, and the repeated DSTK sequences. These structure characteristics are similar to other members of secretogranin family. The human SgIII has homologous proteins in mouse, rat, and Xenopus laevis. Genomic organization shows the gene includes 12 coding exons spanning 39 kb of genomic DNA on the human chromosome 15. Human SgIII is expressed widely as showed in Northern blot and its cDNA hybridizes to 2.2 kb and 1.9 kb bands in many tissues, with two additional 4.5 kb and 3.3 kb bands in brain. Subcelluar localization and immunoblotting indicated SgIII was secreted out of cell through trans-Golgi network (TGN). SgIII may take effect in the biogenesis of secretory granules as a helper protein and be involved in the production or release of peptide hormones in the regulated secretory pathway.