Delocalized quinolinium-macrocyclic peptides, an atypical chemotype for CNS penetration.

Macrocyclic drugs can address an increasing range of molecular targets but enabling central nervous system (CNS) access to these drugs has been viewed as an intractable problem. We designed and synthesized a series of quinolinium-modified cyclosporine derivatives targeted to the mitochondrial cyclophilin D protein. Modification of the cation to enable greater delocalization was confirmed by x-ray crystallography of the cations. Critically, greater delocalization improved brain concentrations. Assessment of the compounds in preclinical assays and for pharmacokinetics identified a molecule JP1-138 with at least 20 times the brain levels of a non-delocalized compound or those reported for cyclosporine. Levels were maintained over 24 hours together with low hERG potential. The paradigm outlined here could have widespread utility in the treatment of CNS diseases.

Therapeutic Validation of GEF-H1 Using a De Novo Designed Inhibitor in Models of Retinal Disease.

Inflammation and fibrosis are important components of diseases that contribute to the malfunction of epithelia and endothelia. The Rho guanine nucleotide exchange factor (GEF) GEF-H1/ARHGEF-2 is induced in disease and stimulates inflammatory and fibrotic processes, cell migration, and metastasis. Here, we have generated peptide inhibitors to block the function of GEF-H1. Inhibitors were designed using a structural in silico approach or by isolating an inhibitory sequence from the autoregulatory C-terminal domain. Candidate inhibitors were tested for their ability to block RhoA/GEF-H1 binding in vitro, and their potency and specificity in cell-based assays. Successful inhibitors were then evaluated in models of TGFß-induced fibrosis, LPS-stimulated endothelial cell-cell junction disruption, and cell migration. Finally, the most potent inhibitor was successfully tested in an experimental retinal disease mouse model, in which it inhibited blood vessel leakage and ameliorated retinal inflammation when treatment was initiated after disease diagnosis. Thus, an antagonist that blocks GEF-H1 signaling effectively inhibits disease features in in vitro and in vivo disease models, demonstrating that GEF-H1 is an effective therapeutic target and establishing a new therapeutic approach.

VEGFA, B, C: Implications of the C-Terminal Sequence Variations for the Interaction with Neuropilins.

Vascular endothelial growth factors (VEGFs) are the key regulators of blood and lymphatic vessels' formation and function. Each of the proteins from the homologous family VEGFA, VEGFB, VEGFC and VEGFD employs a core cysteine-knot structural domain for the specific interaction with one or more of the cognate tyrosine kinase receptors. Additional diversity is exhibited by the involvement of neuropilins-transmembrane co-receptors, whose b1 domain contains the binding site for the C-terminal sequence of VEGFs. Although all relevant isoforms of VEGFs that interact with neuropilins contain the required C-terminal Arg residue, there is selectivity of neuropilins and VEGF receptors for the VEGF proteins, which is reflected in the physiological roles that they mediate. To decipher the contribution made by the C-terminal sequences of the individual VEGF proteins to that functional differentiation, we determined structures of molecular complexes of neuropilins and VEGF-derived peptides and examined binding interactions for all neuropilin-VEGF pairs experimentally and computationally. While X-ray crystal structures and ligand-binding experiments highlighted similarities between the ligands, the molecular dynamics simulations uncovered conformational preferences of VEGF-derived peptides beyond the C-terminal arginine that contribute to the ligand selectivity of neuropilins. The implications for the design of the selective antagonists of neuropilins' functions are discussed.

Peptides Derived from Vascular Endothelial Growth Factor B Show Potent Binding to Neuropilin-1.

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.

Hepatitis C virus exploits cyclophilin A to evade PKR.

Counteracting innate immunity is essential for successful viral replication. Host cyclophilins (Cyps) have been implicated in viral evasion of host antiviral responses, although the mechanisms are still unclear. Here, we show that hepatitis C virus (HCV) co-opts the host protein CypA to aid evasion of antiviral responses dependent on the effector protein kinase R (PKR). Pharmacological inhibition of CypA rescues PKR from antagonism by HCV NS5A, leading to activation of an interferon regulatory factor-1 (IRF1)-driven cell intrinsic antiviral program that inhibits viral replication. These findings further the understanding of the complexity of Cyp-virus interactions, provide mechanistic insight into the remarkably broad antiviral spectrum of Cyp inhibitors, and uncover novel aspects of PKR activity and regulation. Collectively, our study identifies a novel antiviral mechanism that harnesses cellular antiviral immunity to suppress viral replication.

Emerging small-molecule treatments for multiple sclerosis: focus on B cells.

Multiple sclerosis (MS) is a major cause of disability in young adults. Following an unknown trigger (or triggers), the immune system attacks the myelin sheath surrounding axons, leading to progressive nerve cell death. Antibodies and small-molecule drugs directed against B cells have demonstrated good efficacy in slowing progression of the disease. This review focusses on small-molecule drugs that can affect B-cell biology and may have utility in disease management. The risk genes for MS are examined from the drug target perspective. Existing small-molecule therapies for MS with B-cell actions together with new drugs in development are described. The potential for experimental molecules with B-cell effects is also considered. Small molecules can have diverse actions on B cells and be cytotoxic, anti-inflammatory and anti-viral. The current B cell-directed therapies often kill B-cell subsets, which can be effective but lead to side effects and toxicity. A deeper understanding of B-cell biology and the effect on MS disease should lead to new drugs with better selectivity, efficacy, and an improved safety profile. Small-molecule drugs, once the patent term has expired, provide a uniquely sustainable form of healthcare.

Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells.

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.

Small Molecule Neuropilin-1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFß) Production in Regulatory T-Cells.

We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFß production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.

Architecture and hydration of the arginine-binding site of neuropilin-1.

Neuropilin-1 (NRP1) is a transmembrane co-receptor involved in binding interactions with variety of ligands and receptors, including receptor tyrosine kinases. Expression of NRP1 in several cancers correlates with cancer stages and poor prognosis. Thus, NRP1 has been considered a therapeutic target and is the focus of multiple drug discovery initiatives. Vascular endothelial growth factor (VEGF) binds to the b1 domain of NRP1 through interactions between the C-terminal arginine of VEGF and residues in the NRP1-binding site including Tyr297, Tyr353, Asp320, Ser346 and Thr349. We obtained several complexes of the synthetic ligands and the NRP1-b1 domain and used X-ray crystallography and computational methods to analyse atomic details and hydration profile of this binding site. We observed side chain flexibility for Tyr297 and Asp320 in the six new high-resolution crystal structures of arginine analogues bound to NRP1. In addition, we identified conserved water molecules in binding site regions which can be targeted for drug design. The computational prediction of the VEGF ligand-binding site hydration map of NRP1 was in agreement with the experimentally derived, conserved hydration structure. Displacement of certain conserved water molecules by a ligand's functional groups may contribute to binding affinity, whilst other water molecules perform as protein-ligand bridges. Our report provides a comprehensive description of the binding site for the peptidic ligands' C-terminal arginines in the b1 domain of NRP1, highlights the importance of conserved structural waters in drug design and validates the utility of the computational hydration map prediction method in the context of neuropilin. DATABASE: The structures were deposited to the PDB with accession numbers PDB ID: 5IJR, 5IYY, 5JHK, 5J1X, 5JGQ, 5JGI.

IKKγ-Mimetic Peptides Block the Resistance to Apoptosis Associated with Kaposi's Sarcoma-Associated Herpesvirus Infection.

Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-κB pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the IκB kinase (IKK) kinase, IKKγ (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIP-IKKγ interaction both in cells and in vitro can be achieved using IKKγ-mimetic peptides. Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-κB pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKKγ. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKKγ is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKKγ complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions.