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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.

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.

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.

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., The Ras/Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways are essential for cancer cell survival. Here, the authors describes a molecule a131 with dual-inhibitory properties, which targets PI5P4K and mitosis, and it is involved in Ras/Raf/MEK/ERK and PI3K/Akt/mTOR crosstalk, thereby causing reversible growth arrest in normal cells and cell death of tumor cells.

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

Methyltransferase enzymes are promising epigenetic oncotargets. Recent efforts toward the development of inhibitors of two methyltransferases, EZH2 and G9a, as potential anticancer therapies are reviewed with a focus on the structure–activity relationships of compounds published from 2012. Benzamide-substituted 2-pyridones are still by far the most popular selective EZH2 inhibitor class but alternative classes are now being reported. There are now three EZH2 inhibitors in clinical development with the first responses in lymphoma patients with tazemetostat. Potent inhibitors of G9a are also published but no examples have yet reached the clinic. Dual blockage of EZH2–G9a is exemplified by one series of compounds. We conclude this review by presenting the three clinical stage compounds with the first clinical response data.

Mutations in leucine‐rich repeat kinase‐2 are the most common cause of familial Parkinson's disease. The prevalent G2019S mutation increase oxidative, kinase and toxic activity and inhibit endogenous peroxidases. We initially screened a library of 84 antioxidants and identified seven phenolic compounds that inhibited kinase activity on leucine‐rich repeat kinase‐2 substrates. The representative antioxidants (piceatannol, thymoquinone, and esculetin) with strong kinase inhibitor activity, reduced loss in dopaminergic neurons, oxidative dysfunction, and locomotor defects in G2019S‐expressing neuronal and Drosophila models compared to weak inhibitors. We provide proof of principle that natural antioxidants with dual antioxidant and kinase inhibitor properties could be useful for leucine‐rich repeat kinase‐2‐linked Parkinson's disease.

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, is100 nM potent against HDACs 2 and 10, submicromolar potent against HDACs 1, 8, and 11, and50-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.

Small molecules offer powerful ways to alter protein function. However, most proteins in the human proteome lack small-molecule probes, including the large class of non-catalytic transmembrane receptors, such as death receptors. We hypothesized that small molecules targeting the interfaces between transmembrane domains (TMDs) in receptor complexes may induce conformational changes that alter receptor function. Applying this concept in a screening assay, we identified a compound targeting the TMD of death receptor p75NTR that induced profound conformational changes and receptor activity. The compound triggered apoptotic cell death dependent on p75NTR and JNK activity in neurons and melanoma cells, and inhibited tumor growth in a melanoma mouse model. Due to their small size and crucial role in receptor activation, TMDs represent attractive targets for small-molecule manipulation of receptor function.

Contemporary chemotherapeutic treatments incorporate the use of several agents in combination. However, selecting the most appropriate drugs for such therapy is not necessarily an easy or straightforward task. Here, we describe a targeted approach that can facilitate the reliable selection of chemotherapeutic drug combinations through the interrogation of drug-resistance gene networks. Our method employed single-cell eukaryote fission yeast (Schizosaccharomyces pombe) as a model of proliferating cells to delineate a drug resistance gene network using a synthetic lethality workflow. Using the results of a previous unbiased screen, we assessed the genetic overlap of doxorubicin with six other drugs harboring varied mechanisms of action. Using this fission yeast model, drug-specific ontological sub-classifications were identified through the computation of relative hypersensitivities. We found that human gastric adenocarcinoma cells can be sensitized to doxorubicin by concomitant treatment with cisplatin, an intra-DNA strand crosslinking agent and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor. Our findings point to the utility of fission yeast as a model and the differential targeting of a conserved gene interaction network when screening for successful chemotherapeutic drug combinations for human cells.

Herein, we describe the synthesis and SAR of a series of small molecule macrocycles that selectively inhibit JAK2 kinase within the JAK family and FLT3 kinase. Following a multiparameter optimization of a key aryl ring of the previously described SB1518 (pacritinib), the highly soluble 14l was selected as the optimal compound. Oral efficacy in the murine collagen-induced arthritis (CIA) model for rheumatoid arthritis (RA) supported 14l as a potential treatment for autoimmune diseases and inflammatory disorders such as psoriasis and RA. Compound 14l (SB1578) was progressed into development and is currently undergoing phase 1 clinical trials in healthy volunteers.

It has previously been reported that hydrogen sulfide (H2S) exerts both pro- and anti-apoptotic activity in different cultured cells [1] , [2] , [3] . However, the precise effect and mechanism(s) involved remain unclear. We have previously reported that the slow-releasing H2S donor, GYY4137 [4] (morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate) exerts anti-cancer activity both in vivo and in vitro [5] . In this report, GYY4137 showed a distinct anti-proliferative effect across a broad range of cancer cell lines but did not affect proliferation in normal cell lines. In contrast, NaHS, which releases large amounts of H2S over a short time course, failed to affect proliferation of either cancer or normal cell lines. This work raised the possibility that H2S donors, with differing H2S-releasing kinetics, may be of value to kill cancer (but not normal) cells. To this end, in preliminary compound screening tests, we examined a number of novel compounds all of which are GYY4137 analogues, for the ability to kill both cancer and normal cell lines. We also determined the H2S-releasing profiles of these compounds using the conventional methylene blue spectrophotometric assay to monitor H2S release. In the course of this study, we identified an additional, slow-releasing H2S donor, FW1010. FW1010 can slow release H2S more than GYY4137 over a few days. Incubation of either FW1010 or GYY4137 (400μM) in culture medium led to the generation of low concentrations of H2S over 6 days. Comparing FW1010 to GYY4137, FW1010 exhibited enhanced, concentration-dependent killing of different human cancer cell lines (e.g. MCF7, HepG2, U2OS, HL-60, MDA-MB-231, SKOV3, and OVCAR10) but had little or only minimal killing effect in a normal cell line (WI38).We propose that the ability of GYY4137 and FW1010 to kill cancer cells in vitro is likely due to their ability to release H2S slowly over a period of several days. The precise mechanism of action is not clear but measuring intracellular pH (pHi) by confocal microscopy we noted that FW1010 treatment caused a significant concentration-dependent, decline in intracellular pH in MCF7 cells and that this effect was greater than that of GYY4137 at identical concentrations. In contrast, neither GYY4137 nor FW1010 affected pHi in a normal cell line (i.e. WI38). Moreover, both GYY4137 and FW1010 significantly increased lactate release and glucose uptake in a concentration dependent manner in MCF7 (but not in W138) cells. FW1010 was more potent than GYY4137 in this regard. These experiments provide further evidence of the anti-cancer effect of GYY4137 in vitro and identify a new analogue of this compound with seemingly greater efficacy. Additional studies are ongoing to define further the possible mechanisms of action of both FW1010 and GYY4137.

Relaxin-3 or insulin-like peptide 7 (INSL7) is the most recently discovered relaxin/insulin-like family peptide. Mature relaxin-3 consists of an A chain and a B chain held by disulphide bonds. According to structure activity relationship studies, the relaxin-3 B chain is more important in binding and activating the receptor. RXFP3 (also known as Relaxin-3 receptor 1, GPCR 135, somatostatin- and angiotensin- like peptide receptor or SALPR) was identified as the cognate receptor for relaxin-3 by expression profiles and binding studies. Recent studies imply roles of this system in mediating stress and anxiety, feeding, metabolism and cognition. Stapling of peptides is a technique used to develop peptide drugs for otherwise undruggable targets. The main advantages of stapling include, increased activity due to reduced proteolysis, increased affinity to receptors and increased cell permeability. Stable agonists and antagonists of RXFP3 are crucial for understanding the physiological significance of this system. So far, agonists and antagonists of RXFP3 are peptides. In this study, for the first time, we have introduced stapling of the relaxin-3 B chain at 14th and 18th positions (14s18) and 18th and 22nd position (18s22). These stapled peptides showed greater helicity than the unstapled relaxin-3 B chain in circular dichroism analysis. Both stapled peptides bound RXFP3 and activated RXFP3 as observed in an inhibition of forskolin-induced cAMP assay and a ERK1/2 activation assay, although with different potencies. Therefore, we conclude that stapling of the relaxin3 B chain does not compromise its ability to activate RXFP3 and is a promising method for developing stable peptide agonists and antagonists of RXFP3 to aid relaxin-3/RXFP3 research.