Predicting Tacrolimus Concentrations in the Skin of Adult Kidney Transplant Recipients: A Feasibility Study.

Solid organ transplant recipients are at an increased risk of developing skin cancers due to chronic immunosuppression, particularly with calcineurin inhibitors. Tacrolimus is the most prescribed calcineurin inhibitor in this patient cohort, and understanding tacrolimus concentrations in the skin will facilitate the development of anti-cancer preventive and therapeutic strategies. Here, we show that in mice, tacrolimus blood levels peaked rapidly ∼1 h post last oral dose while skin levels rose more slowly and remained high for at least 6 h. Subsequently, tacrolimus skin and blood concentrations were assessed in 15 kidney transplant recipients. The mean age was 61 years, the average time post-transplant was 7 years (range 0-21 years) and 87% were male. The average skin sampling time post tacrolimus dosing was 6 h 32 min. Skin tacrolimus concentrations ranged from 7.1 ng/g to 71.2 ng/g and correlated with blood concentrations (r = 0.6). Mouse and human mean skin concentrations were in a similar range. Our data suggests that tacrolimus measurements in the blood may be used to approximate tacrolimus concentrations in the skin of kidney transplant recipients, and further exploited for the delivery of anti-cancer therapies designed to antagonize the immunosuppressive effects of tacrolimus in the skin.

o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2.

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Prodrugs of the cancer cell selective anti-cancer agent (Z)-2-(1H-indol-3-yl)-3-(isoquinolin-5-yl)acrylonitrile (A131) are orally efficacious in a mouse model of resistant colon cancer.

A131 (1) possesses a unique cancer cell selective dual mechanism of action where cancer cells are killed but normal cells only undergo growth arrest and are able to regrow after removal of 1. SAR studies of 1 indicate that only the specific structure of 1 elicits the full pharmacological effect. However, application of 1 in mouse models of cancer has been hampered by its low solubility and stability when given orally. In this work we describe the study of various prodrugs based on modification of the indole nitrogen. A range of acyl analogues were prepared as prodrugs which were shown to undergo degradation to the parent drug in plasma. A preferred prodrug fully elicited the pharmacological effects of 1 in cells and led to high aqueous solubility suitable for oral administration. In a mouse model of paclitaxel-resistant colon cancer, compound 10, as a TFA salt, showed 76% tumor growth inhibition when administered at an oral dose of 80 mg/kg twice a day.

SAHA and cisplatin sensitize gastric cancer cells to doxorubicin by induction of DNA damage, apoptosis and perturbation of AMPK-mTOR signalling.

Chemotherapy remains the most prescribed anti-cancer therapy, despite patients suffering severe side effects and frequently developing chemoresistance. These complications can be partially overcome by combining different chemotherapeutic agents that target multiple biological pathways. However, selecting efficacious drug combinations remains challenging. We previously used fission yeast Schizosaccharomycespombe as a surrogate model to predict drug combinations, and showed that suberoylanilide hydroxamic acid (SAHA) and cisplatin can sensitise gastric adenocarcinoma cells toward the cytotoxic effects of doxorubicin. Yet, how this combination undermines cell viability is unknown. Here, we show that SAHA and doxorubicin markedly enhance the cleavage of two apoptosis markers, caspase 3 and poly-ADP ribose polymerase (PARP-1), and increase the phosphorylation of γH2AX, a marker of DNA damage. Further, we found a prominent reduction in Ser485 phosphorylation of AMP-dependent protein kinase (AMPK), and reductions in its target mTOR and downstream ribosomal protein S6 phosphorylation. We show that SAHA contributes most of the effect, as confirmed using another histone deacetylase inhibitor, trichostatin A. Overall, our results show that the combination of SAHA and doxorubicin can induce apoptosis in gastric adenocarcinoma in a synthetically lethal manner, and that fission yeast offers an efficient tool for identifying potent drug combinations against human cancer cells.

Design and synthesis of triple inhibitors of janus kinase (JAK), histone deacetylase (HDAC) and Heat Shock Protein 90 (HSP90).

Inhibition of multiple signaling pathways in a cancer cell with a single molecule could result in better therapies that are simpler to administer. Efficacy may be achieved with reduced potency against individual targets if there is synergy through multiple pathway inhibition. To achieve this, it is necessary to be able to build multi-component ligands by joining together key pharmacophores in a way which maintains sufficient activity against the individual pathways. In this work, designed triple inhibiting ligands are explored aiming to block three completely different target types: a kinase (JAK2), an epigenetic target (HDAC) and a chaperone (HSP90). Although these enzymes have totally different functions they are related through inter-dependent pathways in the developing cancer cell. Synthesis of several complex multi-inhibiting ligands are presented along with initial enzyme inhibition data against 3 biological target classes of interest. A lead compound, 47, was discovered which had low micromolar activity for all 3 targets. Further development of these complex trispecific designed multiple ligands could result in a 'transient drug', an alternative combination therapy for treating cancer mediated via a single molecule.

Merging of ruxolitinib and vorinostat leads to highly potent inhibitors of JAK2 and histone deacetylase 6 (HDAC6).

Inhibition of more than one pathway in a cancer cell with a single molecule could result in better therapies with less complex dosing regimens. In this work multi-component ligands have been prepared by joining together key pharmacophores of two different enzyme inhibitors in a way which increases potency against the individual pathways. Selective JAK1/2 inhibitor, ruxolitinib (3), and pan-HDAC inhibitor vorinostat (4) were linked together by a single nitrogen atom to create a new series of compounds with very potent JAK2 and HDAC6 inhibition with selectivity against HDAC1. A preferred compound, 13b, had unprecedented sub-nanomolar JAK2 potency with an IC50 of 41 pM and a sub-nanomolar IC50 against HDAC6 of 200 pM. Binding models show a good fit into both JAK2 and HDAC6.

Towards Selective Mycobacterial ClpP1P2 Inhibitors with Reduced Activity against the Human Proteasome.

Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti-M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti-M. tuberculosis activity.

Discovery of medium ring thiophosphorus based heterocycles as antiproliferative agents.

Hydrogen sulfide (H2S) has been investigated for its potential in therapy. Recently, we reported novel H2S donor molecules based on a thiophosphorus core, which slowly release H2S and have improved anti-proliferative activity in cancer cell lines compared to the most widely studied H2S donor GYY4137 (1). Herein, we have prepared new thiophosphorus organic H2S donors with different ring sizes and evaluated them in two solid tumor cell lines and one normal cell line. A seven membered ring compound, 17, was found to be the most potent with sub-micromolar IC50s in breast (0.76µM) and ovarian (0.76µM) cancer cell lines. No significant H2S release was detected in aqueous solution for this compound. However, confocal imaging showed that H2S was released from 17 inside cells at a similar level to the widely studied H2S donor GYY4137, which was shown to release 10µM H2S after 12h at a concentration of 400µM. Comparison of 17 with its non-sulfur oxygen analogue, 26, provided evidence that the sulfur atom is important for its potency. However, the significant potency observed for 26 (5.94-11.0µM) indicates that the high potency of 17 is not entirely due to release of H2S. Additional mechanism(s) appear to be responsible for the observed activity, hence more detailed studies are required to better understand the role of H2S in cancer with potent thiophosphorus agents.

Overcoming Chloroquine Resistance in Malaria: Design, Synthesis, and Structure-Activity Relationships of Novel Hybrid Compounds.

Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge. Reversal of acquired resistance can be achieved using agents that resensitize resistant parasites to a previously efficacious therapy. Building on our initial work describing novel chemoreversal agents (CRAs) that resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA scaffold to chloroquine produces hybrid compounds with restored potency toward a range of resistant malaria parasites. A preferred compound, compound 35, showed broad activity and good potency against seven strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane permeability, and in vitro toxicity in a hepatocyte line and a cardiomyocyte line indicates that compound 35 has a good therapeutic window and favorable drug-like properties. This study provides initial support for CQ-CRA hybrid compounds as a potential treatment for resistant malaria.

A novel slow-releasing hydrogen sulfide donor, FW1256, exerts anti-inflammatory effects in mouse macrophages and in vivo.

Exogenous hydrogen sulfide (H2S) is known to exert anti-inflammatory effects both in macrophages and in animal models. In this study, we first showed that NaHS caused a concentration dependent reduction in TNFα and IL-6 secretion in LPS-stimulated RAW264.7 macrophages in the absence of cell death. Thereafter, we screened a series of novel slow H2S donors for similar activity. One such compound, FW1256, concentration dependently decreased TNFα, IL-6, PGE2 and NO generation in LPS-stimulated RAW264.7 macrophages and BMDMs. FW1256 also significantly reduced IL-1ß, COX-2 and iNOS mRNA and protein in LPS-stimulated RAW264.7 macrophages. Mechanistically, FW1256 decreased NFκB activation as evidenced by reduced cytosolic phospho-IκBα levels and reduced nuclear p65 levels in LPS-stimulated RAW264.7 macrophages treated with FW1256. Using a H2S fluorescent probe in FW1256-treated RAW264.7 macrophages, H2S release from FW1256 was apparent over a period of 24h in these cells. Moreover, the effect of FW1256 on TNFα and IL-6 by FW1256 in LPS-stimulated RAW264.7 macrophages was reversed by treatment with the H2S scavenger, vitamin B12a. FW1256 had no cytotoxic effect on LPS-stimulated RAW264.7 macrophages or BMDMs. In vivo, FW1256 administration also reduced IL-1ß, TNFα, nitrate/nitrite and PGE2 levels in LPS-treated mice. We show here a novel slow H2S-releasing compound that exerts anti-inflammatory effects in macrophages and in vivo. FW1256 may be a useful tool to study the biological effects of exogenous H2S and could also have future therapeutic value in inflammatory conditions.

Fluorescent Probes for H2S Detection and Quantification.

Many diverse, sensitive and structurally novel fluorescent probes have recently been reported for H2S detection. Quantification of H2S requires a selective chemosensor which will react only with H2S against a background of high concentrations of other thiols or reducing agents. Most published probes are able to quantify H2S selectively in a simple in vitro system with the most sensitive probes able to detect H2S at below 100 nM concentrations. A subset of probes also have utility in sensing H2S in living cells, and there are now several with specific sub-cellular localization and a few cases of in vivo applications. Biologists studying H2S now have a wide range of tools to assist them to aid further understanding of the role of H2S in biology.

Structure and ligand-based design of mTOR and PI3-kinase inhibitors leading to the clinical candidates VS-5584 (SB2343) and SB2602.

Phosphoinositide 3-kinases (PI3Ks) and the mammalian target of rapamycin (mTOR) act as critical effectors in a commonly deregulated cell signaling pathway in human cancers. The abnormal activation of the PI3K/mTOR pathway has been shown to play a role in initiation, progression, and metastasis of human tumors. Being one of the most frequently activated pathways in cancer, much effort has been directed toward inhibition of the PI3K/mTOR pathway as a novel oncology therapy. Previous work by a number of groups has revealed several selective PI3K and dual mTOR/PI3K inhibitors. However, there are few reports of therapeutic agents with a pan-PI3K/mTOR inhibitory profile within a narrow concentration range. We therefore initiated a drug discovery project with the aim of discovering dual mTOR/PI3K inhibitors which would equipotently inhibit the 4 isoforms of PI3K, α, ß, γ, and δ, and mTOR a compelling profile for powerful blockage of the PI3K/mTOR pathway. A pharmacophore model was generated and used for designing a series of novel compounds, based on a purine scaffold, which potently inhibited mTOR and PI3Ks. These compounds contained a phenol headgroup essential for binding to the target proteins. Early efforts concentrated on finding replacements for the phenol as it was rapidly conjugated resulting in a short half-life in vivo. Compounds with a variety of headgroups were docked into the PI3Kα and mTOR ATP-binding sites, and aminopyrimidine and aminopyrazine were found to make excellent phenol replacements. Further structure guided optimization of side chains in the 8- and 9-positions of the purine resulted in potent inhibitors with good PKDM properties. As the PI3 kinases play a role in insulin signaling, it is believed that targeting mTOR selectively may give the benefit of blocking the AKT-pathway while avoiding the potential side effects associated with PI3K inhibition. As a result we designed a further series of selective mTOR kinase inhibitors. The project was successfully concluded by progressing both a dual mTOR/PI3K inhibitor, SB2343, and a selective mTOR inhibitor, SB2602, into preclinical development. SB2343 has since entered phase 1 clinical development as VS-5584.

SB1578, a novel inhibitor of JAK2, FLT3, and c-Fms for the treatment of rheumatoid arthritis.

SB1578 is a novel, orally bioavailable JAK2 inhibitor with specificity for JAK2 within the JAK family and also potent activity against FLT3 and c-Fms. These three tyrosine kinases play a pivotal role in activation of pathways that underlie the pathogenesis of rheumatoid arthritis. SB1578 blocks the activation of these kinases and their downstream signaling in pertinent cells, leading to inhibition of pathological cellular responses. The biochemical and cellular activities of SB1578 translate into its high efficacy in two rodent models of arthritis. SB1578 not only prevents the onset of arthritis but is also potent in treating established disease in collagen-induced arthritis mice with beneficial effects on histopathological parameters of bone resorption and cartilage damage. SB1578 abrogates the inflammatory response and prevents the infiltration of macrophages and neutrophils into affected joints. It also leads to inhibition of Ag-presenting dendritic cells and inhibits the autoimmune component of the disease. In summary, SB1578 has a unique kinase spectrum, and its pharmacological profile provides a strong rationale for the ongoing clinical development in autoimmune diseases.

Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

BACKGROUND: Immunosuppressive drugs such as tacrolimus have revolutionized our ability to transplant organs between individuals. Tacrolimus acts systemically to suppress the activity of T-cells within and around transplanted organs. However, tacrolimus also suppresses T-cell function in the skin, contributing to a high incidence of skin cancer and associated mortality and morbidity in solid organ transplant recipients. Here, we aimed to identify a compound capable of re-establishing antitumor T-cell control in the skin despite the presence of tacrolimus. METHODS: In this study, we performed time-resolved fluorescence resonance energy transfer to identify molecules capable of antagonizing the interaction between tacrolimus and FKBP12. The capacity of these molecules to rescue mouse and human T-cell function in the presence of tacrolimus was determined in vitro, and the antitumor effect of the lead compound, Q-2361, was assessed in "regressor" models of skin cancer in immunosuppressed mice. Systemic CD8 T-cell depletion and analyses of intratumoral T-cell activation markers and effector molecule production were performed to determine the mechanism of tumor rejection. Pharmacokinetic studies of topically applied Q-2361 were performed to assess skin and systemic drug exposure. RESULTS: Q-2361 potently blocked the interaction between tacrolimus and FKBP12 and reversed the inhibition of the nuclear factor of activated T cells activation by tacrolimus following T-cell receptor engagement in human Jurkat cells. Q-2361 rescued T-cell function in the presence of tacrolimus, rapamycin, and everolimus. Intratumoral injection of Q-2361-induced tumor regression in mice systemically immune suppressed with tacrolimus. Mechanistically, Q-2361 treatment permitted T-cell activation, proliferation, and effector function within tumors. When CD8 T cells were depleted, Q-2361 could not induce tumor regression. A simple solution-based Q-2361 topical formulation achieved high and sustained residence in the skin with negligible drug in the blood. CONCLUSIONS: Our findings demonstrate that the local application of Q-2361 permits T-cells to become activated driving tumor rejection in the presence of tacrolimus. The data presented here suggests that topically applied Q-2361 has great potential for the reactivation of T-cells in the skin but not systemically, and therefore represents a promising strategy to prevent or treat skin malignancies in immunosuppressed organ transplant recipients.

Structure-based design of PDK1 inhibitors.

A macrocyclic 2-anilino-4-phenyl-pyrimidine CDK/Flt3/JAK2 inhibitor was found to have moderate PDK1 activity. After docking into a PDK1 X-ray structure it was suggested that the pyrimidine ring could be substituted for a purine thereby increasing the number of hydrophobic contacts with the protein and forming an additional hydrogen bond to the kinase hinge. Deletion of the macrocyclic linker allowed a more rapid optimisation of the aromatic substituents as well as the introduction of an amino-amide solubility tag. This improved both binding to the enzyme and physiochemical properties without compromising ligand efficiency.

Structure-based optimization of morpholino-triazines as PI3K and mTOR inhibitors.

A virtual screen of our in-house database using various fingerprint techniques returned several triazine hits which were found to be mTOR inhibitors with a slight selectivity over PI3Kα. Using structure-guided lead optimization the inhibitory activity towards mTOR and PI3Kα was increased to the low nanomolar range. Exploiting shape differences in the binding-site allowed for the design of mTOR selective inhibitors. Focus on ligand efficiency ensured the inhibitors retained a low molecular weight and desirable drug-like properties.

Thieno[3,2-d]pyrimidin-4(3H)-one derivatives as PDK1 inhibitors discovered by fragment-based screening.

Ligand efficient fragments binding to PDK1 were identified by an NMR fragment-based screening approach. Computational modeling of the fragments bound to the active site led to the design and synthesis of a series of novel 6,7-disubstituted thienopyrimidin-4-one compounds, with low micromolar inhibitory activity against PDK1 in a biochemical enzyme assay.

2-anilino-4-aryl-8H-purine derivatives as inhibitors of PDK1.

A series of 2-anilino substituted 4-aryl-8H-purines were prepared as potent inhibitors of PDK1, a serine-threonine kinase thought to play a role in the PI3K/Akt signaling pathway, a key mediator of cancer cell growth, survival and tumorigenesis. The synthesis, SAR and ADME properties of this series of compounds are discussed culminating in the discovery of compound 6 which possessed sub-micromolar cell proliferation activity and 65% oral bioavailability in mice.

Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3).

Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.

DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: synthesis and antibacterial activity.

The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.