ISM1 protects lung homeostasis via cell-surface GRP78-mediated alveolar macrophage apoptosis.

Alveolar macrophages (AMs) are critical for lung immune defense and homeostasis. They are orchestrators of chronic obstructive pulmonary disease (COPD), with their number significantly increased and functions altered in COPD. However, it is unclear how AM number and function are controlled in a healthy lung and if changes in AMs without environmental assault are sufficient to trigger lung inflammation and COPD. We report here that absence of isthmin 1 (ISM1) in mice (Ism1-/- ) leads to increase in both AM number and functional heterogeneity, with enduring lung inflammation, progressive emphysema, and significant lung function decline, phenotypes similar to human COPD. We reveal that ISM1 is a lung resident anti-inflammatory protein that selectively triggers the apoptosis of AMs that harbor high levels of its receptor cell-surface GRP78 (csGRP78). csGRP78 is present at a heterogeneous level in the AMs of a healthy lung, but csGRP78high AMs are expanded in Ism1-/- mice, cigarette smoke (CS)-induced COPD mice, and human COPD lung, making these cells the prime targets of ISM1-mediated apoptosis. We show that csGRP78high AMs mostly express MMP-12, hence proinflammatory. Intratracheal delivery of recombinant ISM1 (rISM1) depleted csGRP78high AMs in both Ism1-/- and CS-induced COPD mice, blocked emphysema development, and preserved lung function. Consistently, ISM1 expression in human lungs positively correlates with AM apoptosis, suggesting similar function of ISM1-csGRP78 in human lungs. Our findings reveal that AM apoptosis regulation is an important physiological mechanism for maintaining lung homeostasis and demonstrate the potential of pulmonary-delivered rISM1 to target csGRP78 as a therapeutic strategy for COPD.

Drysdalin, a snake neurotoxin with higher affinity for soluble acetylcholine binding protein from Aplysia californica than from Lymnaea stagnalis.

Acetylcholine binding proteins (AChBPs), structural and functional surrogates of the extracellular binding domain of nicotinic acetylcholine receptor (nAChRs), in complex with various antagonists and agonists have provided detailed insights into the neurotransmitter binding site of nAChRs. The classical long-chain α-neurotoxins bungarotoxin (44-fold) and cobratoxin (7-fold) bind to Lymnaea stagnalis (Ls)-AChBP with higher affinity compared to Aplysia californica (Ac)-AChBP. In this study, we describe a novel long chain α-neurotoxin Drysdalin, which has higher binding affinity (7-fold) to Ac-AChBP when compared to Ls-AChBP. This suggests an involvement of different regions or modes of interaction of drysdalin, when compared to the bungarotoxin and cobratoxin. We also found that the C-terminal 24-amino acid residues of drysdalin are critical for the binding to Ac-AChBP and its removal caused ~90-fold reduction in affinity. Further to understand the interaction of drysdalin with Ac-AChBP, we studied the role of three non-conserved amino acid residues of drysdalin, namely Arg30, Leu34 and Ala37. Substitution of Arg30 with the conserved Phe residue caused a ~100-fold reduction, Leu34 with conserved Arg caused a ~6-fold reduction, whereas substitution of Ala37 with conserved Arg enhanced the binding by 3-fold. The dramatic influence of this carboxyl terminal sequence enriched in arginine and proline residues suggests that the toxin binding pose is influenced primarily by this extended sequence.

Discovery of small molecules targeting GRP78 for antiangiogenic and anticancer therapy.

Glucose Regulated Protein 78 kDa (GRP78) is an attractive antiangiogenic and anticancer target for its selective accumulation on the surface of cancer cells and cancer endothelial cells rather than normal cells. In this study, we identified a novel series of small molecules that binds to GRP78, exhibiting potent antiangiogenic and anticancer activities without affecting normal cells. Among these, FL5,2-(4-((4-acetamidophenoxy)methyl)phenyl)-N-isobutylbenzofuran-3-carboxamide, was superior to others due to its strong binding affinity to GRP78 (an increase in the Tm > 2 °C stabilising the GRP78 protein) and potent antiangiogenic and anticancer activities against human umbilical vein endothelial cells (HUVEC) (EC50 = 1.514 µM) and human renal cancer cells (786-O) (50% cell death at 10 µM). Furthermore, FL5 displayed no cytotoxic activity towards mouse fibroblast cells (Swiss-3T3), which do not harbour cell surface GRP78 under normal condition. FL5 was less detrimental to ATPase activity, which is essential for normal cells, as seen in the virtual docking studies. This study reports the discovery of novel small molecules targeting GRP78 with potent antiangiogenic and anticancer activities and less toxicity to normal cells, which provides prototype candidates for novel paths for cancer therapy.

Drysdalin, an antagonist of nicotinic acetylcholine receptors highlights the importance of functional rather than structural conservation of amino acid residues.

Snake venom neurotoxins are potent antagonists of nicotinic acetylcholine receptors (nAChRs). Here, we describe a novel member of class 3c long-chain neurotoxin drysdalin from the venom of Drysdalia coronoides. Drysdalin lacks three of the eight conserved classical functional residues critical for nAChRs interaction. Despite such a drastic alteration of the functional site, recombinant drysdalin showed irreversible postsynaptic neurotoxicity with nanomolar potency and selectively antagonizes the rodent muscle (α1)2ß1δε, and human α7 and α9α10 nAChRs, but had no significant activity at the human α3ß2, α3ß4, α4ß2, and α4ß4 nAChRs. Substitution of Leu34 and Ala37 residues with the conserved Arg had minimal impact on the potency whereas conserved Phe replacement of residue Arg30 substantially reduced or abolished inhibitory activity. In contrast, truncation of the 24-residue long C-terminal tail leads to complete loss in (a) activity at α9α10 nAChR; and (b) irreversibility with reduced potency at the muscle and α7 nAChRs. Overall, the non-conserved Arg30 residue together with the uniquely long C-terminal tail contribute to the inhibitory activity of drysdalin at the nAChRs suggesting, at least for drysdalin, functional rather than sequence conservation plays a critical role in determining the activity of the toxin.

Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice.

Glucose regulated protein 78 kDa (GRP78) is a recently emerged target for cancer therapy and a biomarker for cancer prognosis. Overexpression of GRP78 is observed in many types of cancers, with the cell-surface GRP78 being preferentially present in cancer cells and cancer blood vessel endothelial cells. Isthmin (ISM) is a secreted high-affinity proapoptotic protein ligand of cell-surface GRP78 that suppresses angiogenesis and tumor growth in mice. The C-terminal AMOP (adhesion-associated domain in MUC4 and other proteins) domain of ISM is critical in mediating its interaction with human umbilical vein endothelial cells (HUVECs). In this work, we report novel cyclic peptides harboring the RKD motif in the ISM AMOP domain that function as proapoptotic ligands of cell-surface GRP78. The most potent peptide, BC71, binds to GRP78 and converge to tumor in mice. Intravenous administration of BC71 suppressed xenograft tumor growth in mice as a single agent, with significant reduction in tumor angiogenesis and upsurge in apoptosis. Fluorescent-labeled BC71 accumulates in tumor in mice by targeting cell-surface GRP78. We show that BC71 triggers apoptosis via cell-surface GRP78 and activates caspase-8 and p53 signaling pathways in HUVECs. Using amide hydrogen-deuterium exchange mass spectrometry (HDXMS), we identified that BC71 preferentially binds to ATP-bound GRP78 via amino acid residues 244-257 of GRP78. Hence, BC71 serves as a valuable prototype for further development of peptidomimetic anticancer drugs targeting cell-surface GRP78 as well as PET imaging agents for cancer prognosis.

Angio-3, a 10-residue peptide derived from human plasminogen kringle 3, suppresses tumor growth in mice via impeding both angiogenesis and vascular permeability.

Anti-angiogenesis therapy is an established therapeutic strategy for cancer. The endogenous angiogenic inhibitor angiostatin contains the first 3-4 kringle domains of plasminogen and inhibits both angiogenesis and vascular permeability. We present here a 10-residue peptide, Angio-3, derived from plasminogen kringle 3, which retains the functions of angiostatin in inhibiting both angiogenesis and vascular permeability. NMR studies indicate that Angio-3 holds a solution structure similar to the corresponding region of kringle 3. Mechanistically, Angio-3 inhibited both VEGF- and bFGF-induced angiogenesis by inhibiting EC proliferation and migration while inducing apoptosis. Inhibition of VEGF-induced vascular permeability results from its ability to impede VEGF-induced dissociation of adherens junction and tight junction proteins as well as the formation of actin stress fibers. When administered intravenously, Angio-3 inhibited subcutaneous breast cancer and melanoma growth by suppressing both tumor angiogenesis and intra-tumor vascular permeability. Hence, Angio-3 is a novel dual inhibitor of angiogenesis and vascular permeability. It is valuable as a lead peptide that can be further developed as therapeutics for diseases involving excessive angiogenesis and/or vascular permeability.