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.

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.

Discovery of a potent histone deacetylase (HDAC) 3/6 selective dual inhibitor.

Herein, we report the discovery of a dual histone deacetylase inhibitor displaying a unique HDAC3/6 selectivity profile. An initial strategy to merge two epigenetic pharmacophores resulted in the discovery of potent HDAC6 inhibitors with selectivity over HDAC1. Screening in an HDAC panel revealed additional low nanomolar inhibition only against HDAC3. Low micromolar antiproliferative activities against two breast cancer and four hematological cancer cell lines was supported by pharmacodynamic studies on a preferred molecule, 24c, substantiating the HDAC inhibitory profile in cells. Apoptosis was identified as one of the main cell death pathways. Modelling studies of 24c against HDAC1,2,3 and 6 further provided insights on the orientation of specific residues relevant to compound potency, explaining the observed HDAC3/6 selectivity. A subset of the compounds also exhibited good antimalarial activities, particularly against the chloroquine-resistant strain K1 of P.falciparum. In vitro studies revealed a favourable DMPK profile warranting further investigation of the therapeutic potential of these compounds.

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.

Design and synthesis of potent dual inhibitors of JAK2 and HDAC based on fusing the pharmacophores of XL019 and vorinostat.

Specifically blocking more than one oncogenic pathway simultaneously in a cancer cell with a combination of different drugs is the mainstay of the majority of cancer treatments. Being able to do this via two targeted pathways without inducing side effects through a general mechanism, such as chemotherapy, could bring benefit to patients. In this work we describe a new dual inhibitor of the JAK-STAT and HDAC pathways through designing and developing two types of molecule based on the JAK2 selective inhibitor XL019 and the pan-HDAC inhibitor, vorinostat. Both series of compounds had examples with low nanomolar JAK2 and HDAC1/6 inhibition. In some cases good HDAC1 selectivity was achieved while retaining HDAC6 activity. The observed potency is explained through molecular docking studies of all three enzymes. One example, 69c had 16-25 fold selectivity against the three other JAK-family proteins JAK1, JAK3 and TYK2. A number of compounds had sub-micromolar potencies against a panel of 4 solid tumor cell lines and 4 hematological cell lines with the most potent compound, 45h, having a cellular IC50 of 70 nM against the multiple myeloma cell line KMS-12-BM. Evidence of both JAK and HDAC pathway inhibition is presented in Hela cells showing that both pathways are modulated. Evidence of apoptosis with two compounds in 4 sold tumor cell lines is also presented.

Discovery of the cancer cell selective dual acting anti-cancer agent (Z)-2-(1H-indol-3-yl)-3-(isoquinolin-5-yl)acrylonitrile (A131).

Selective targeting of cancer cells over normal cells is a key objective of targeted therapy. However few approaches achieve true mechanistic selectivity resulting in debilitating side effects and dose limitation. In this work we describe the discovery of A131 (4a), a new agent with an unprecedented dual mechanism of action targeting both mitosis and autophagy. Compound 4a was first identified in a phenotypic screen in which HeLa cells treated with 4a manifested mitotic arrest along with formation of multiple vesicles. Further investigations showed that 4a causes an increase in mitotic marker pH3 and autophagy marker LC3. Importantly 4a induces cell death in cancer cells while sparing normal cells which regrow after 4a is removed. Dual activities against pH3 and LC3 markers are required for cancer cell selectivity. An extensive SAR investigation confirmed 4a as the optimal dual inhibitor with potency against a panel of 30 cancer cell lines (average antiproliferative GI50 1.5 µM). In a mouse model of paclitaxel-resistant colon cancer, 4a showed 74% tumor growth inhibition when administered at a dose of 20 mg/kg IP 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.

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.

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.

Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality.

Achieving robust cancer-specific lethality is the ultimate clinical goal. Here, we identify a compound with dual-inhibitory properties, named a131, that selectively kills cancer cells, while protecting normal cells. Through an unbiased CETSA screen, we identify the PIP4K lipid kinases as the target of a131. Ablation of the PIP4Ks generates a phenocopy of the pharmacological effects of PIP4K inhibition by a131. Notably, PIP4Ks inhibition by a131 causes reversible growth arrest in normal cells by transcriptionally upregulating PIK3IP1, a suppressor of the PI3K/Akt/mTOR pathway. Strikingly, Ras activation overrides a131-induced PIK3IP1 upregulation and activates the PI3K/Akt/mTOR pathway. Consequently, Ras-transformed cells override a131-induced growth arrest and enter mitosis where a131's ability to de-cluster supernumerary centrosomes in cancer cells eliminates Ras-activated cells through mitotic catastrophe. Our discovery of drugs with a dual-inhibitory mechanism provides a unique pharmacological strategy against cancer and evidence of cross-activation between the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways via a Ras˧PIK3IP1˧PI3K signaling network.

Intracellular Hyper-Acidification Potentiated by Hydrogen Sulfide Mediates Invasive and Therapy Resistant Cancer Cell Death.

Slow and continuous release of H2S by GYY4137 has previously been demonstrated to kill cancer cells by increasing glycolysis and impairing anion exchanger and sodium/proton exchanger activity. This action is specific for cancer cells. The resulting lactate overproduction and defective pH homeostasis bring about intracellular acidification-induced cancer cell death. The present study investigated the potency of H2S released by GYY4137 against invasive and radio- as well as chemo-resistant cancers, known to be glycolytically active. We characterized and utilized cancer cell line pairs of various organ origins, based on their aggressive behaviors, and assessed their response to GYY4137. We compared glycolytic activity, via lactate production, and intracellular pH of each cancer cell line pair after exposure to H2S. Invasive and therapy resistant cancers, collectively termed aggressive cancers, are receptive to H2S-mediated cytotoxicity, albeit at a higher concentration of GYY4137 donor. While lactate production was enhanced, intracellular pH of aggressive cancers was only modestly decreased. Inherently, the magnitude of intracellular pH decrease is a key determinant for cancer cell sensitivity to H2S. We demonstrated the utility of coupling GYY4137 with either simvastatin, known to inhibit monocarboxylate transporter 4 (MCT4), or metformin, to further boost glycolysis, in bringing about cell death for aggressive cancers. Simvastatin inhibiting lactate extrusion thence contained excess lactate induced by GYY4137 within intracellular compartment. In contrast, the combined exposure to both GYY4137 and metformin overwhelms cancer cells with lactate over-production exceeding its expulsion rate. Together, GYY4137 and simvastatin or metformin synergize to induce intracellular hyper-acidification-mediated cancer cell death.

Design and Synthesis of Ligand Efficient Dual Inhibitors of Janus Kinase (JAK) and Histone Deacetylase (HDAC) Based on Ruxolitinib and Vorinostat.

Concomitant inhibition of multiple oncogenic pathways is a desirable goal in cancer therapy. To achieve such an outcome with a single molecule would simplify treatment regimes. Herein the core features of ruxolitinib (1), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (2), leading to new molecules that are bispecific targeted JAK/HDAC inhibitors. A preferred pyrazole substituted pyrrolopyrimidine, 24, inhibits JAK1 and HDACs 1, 2, 3, 6, and 10 with IC50 values of less than 20 nM, is <100 nM potent against JAK2 and HDAC11, and is selective for the JAK family against a panel of 97 kinases. Broad cellular antiproliferative potency of 24 is supported by demonstration of JAK-STAT and HDAC pathway blockade in hematological cell lines. Methyl analogue 45 has an even more selective profile. This study provides new leads for assessment of JAK and HDAC pathway dual inhibiton achieved with a single molecule.

Bortezomib Warhead-Switch Confers Dual Activity against Mycobacterial Caseinolytic Protease and Proteasome and Selectivity against Human Proteasome.

Mycobacteria harbor two main degradative proteolytic machineries, the caseinolytic protease ClpP1P2 and a proteasome. We recently showed that Bortezomib inhibits ClpP1P2 and exhibits whole cell activity against Mycobacterium tuberculosis. Bortezomib, a dipeptide with a boronic acid warhead, is a human proteasome inhibitor approved for cancer therapy. The boronic acid warhead of the compound has been shown to drive potency against both the human proteasome and ClpP1P2 protease. Selectivity for the bacterial ClpP1P2 protease over the human proteasome is lacking but needs to be achieved to move this new anti-tuberculosis lead forward. In this study we explored whether an alternative warhead could influence Bortezomib's selectivity. We synthesized an analog containing a chloromethyl ketone instead of the boronic acid warhead and determined potencies against the bacterial and human enzymes. Surprisingly, the analog retained activity against mycobacterial ClpP1P2 and was active against the mycobacterial proteasome, but was devoid of activity against the human proteasome. Interrogation of a set of chloromethyl ketone peptides identified three additional compounds similarly inhibiting both ClpP1P2 and the proteasome in the bacteria while leaving the human proteasome untouched. Finally, we showed that these compounds display bactericidal activity against M. tuberculosis with cytotoxicity ranging from acceptable to undetectable. These results suggest that selectivity over the human proteasome is achievable. Selectivity, together with dual-targeting of mycobacterial ClpP1P2 and proteasome makes this new scaffold an attractive starting point for optimization.

Analysis of Protein Target Interactions of Synthetic Mixtures by Affinity-LC/MS.

Analysis of interactions between molecules is of fundamental importance in life science research. In this study, we applied weak affinity chromatography, based on high-performance liquid chromatography and mass spectrometry, as a powerful tool for direct analysis of the components of a chemical reaction mixture for their binding to a target protein. As a demonstration of the potential of this method, we analyzed the binding of the compounds of the reaction mixture to the chaperone heat shock protein 90 (Hsp90). It was possible to analyze quantitatively the binding of the components of the mixture to the target independently from each other without any preceding process such as purification. This feature has wide implications in biological sciences as crude mixtures, either natural or synthetic, can be analyzed directly for their possible binding to a target. This method could lead to savings in costs and labor through shortening chemical research project development time.

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.

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.

Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria.

Reactive multi-target 'fragment drugs' represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small 'dirty' drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM pathogens. These hits will now be evaluated in animal models of mycobacterial infection to determine whether any of them can be moved forward as a new antimycobacterial fragment drug candidate.

Design and Synthesis of Janus Kinase 2 (JAK2) and Histone Deacetlyase (HDAC) Bispecific Inhibitors Based on Pacritinib and Evidence of Dual Pathway Inhibition in Hematological Cell Lines.

Blockage of more than one oncoprotein or pathway is now a standard approach in modern cancer therapy. Multiple inhibition is typically achieved with two or more drugs. Herein, we describe a pharmacophore merging strategy combining the JAK2/FLT3 inhibitor pacritnib with the pan-HDAC inhibitor, vorinostat, to create bispecific single molecules with both JAK and HDAC targeted inhibition. A preferred ether hydroxamate, 51, inhibits JAK2 and HDAC6 with low nanomolar potency, is <100 nM potent against HDACs 2 and 10, submicromolar potent against HDACs 1, 8, and 11, and >50-fold selective for JAK2 in a panel of 97 kinases. Broad cellular antiproliferative potency is supported by demonstration of JAK-STAT and HDAC pathway blockade in several hematological cell lines, inhibition of colony formation in HEL cells, and analysis of apoptosis. This study provides new tool compounds for further exploration of dual JAK-HDAC pathway inhibiton achieved with a single molecule.