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Deconstruction of a bicyclic tetrahydroquinoline-6-carboxylic acid scaffold into aminosalicylic acids yielded dual MCL-1/BCL-xL inhibitors from an MCL-1 selective lead.

The disruption of aberrant protein-protein interactions (PPIs) with synthetic agents remains a challenging goal in contemporary medicinal chemistry but some progress has been made. One such dysregulated PPI is that between the anti-apoptotic Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and the α-helical Bcl-2 homology-3 (BH3) domains of its pro-apoptotic counterparts, such as Bak. Herein, we describe the discovery of small-molecule inhibitors of the Mcl-1 oncoprotein based on a novel chemotype. Particularly, re-engineering of our α-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates afforded inhibitors of comparable potencies but with significantly decreased molecular weights. The most potent inhibitor 2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic acid (1 r: Ki =2.90 μm) likely binds in the p2 pocket of Mcl-1 and engages R263 in a salt bridge through its carboxylic acid, as supported by 2D (1) H-(15) N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts.

Mimicry of two faces of an α-helix might yield more potent and more selective inhibitors of dysregulated, helix-mediated protein–protein interactions (PPI). Herein, we demonstrate that a 2,6,9-tri-substituted purine is capable of disrupting the Mcl-1–Bak-BH3 PPI through effective mimicry of key residues on opposing faces of the Bak-BH3 α-helix.

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 and p3 pockets of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Molecular modeling using the Site-Identification by Ligand Competitive Saturation (SILCS) approach was used to qualitatively direct ligand design as well as develop quantitative models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions with the p2 pockets dominate the affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 20-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 versus Bcl-xL. The SILCS-based SAR of the present compounds represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumours and hematological cancers, including acute myeloid leukaemia.

3-Substituted, 2,6-dichloropyridines have featured in the syntheses of small molecule inhibitors of a wide variety of biological targets. Hence, the regioselective displacement of the chlorines is of significant interest. Through conducting an extensive solvent study, we have found that non-polar, aprotic solvents of low hydrogen bond basicities favour substitution of the chlorine ortho to the 3-substituent by alkali metal alkoxides. We present convincing evidence that coordination of the alkali metal counter-ion to the 3-substituent (nitro, ester, amide) is the origin of the ortho-selectivity to give a cyclic, six-membered transition state. Excellent ortho-selectivities (⩾98:2) for secondary and tertiary alkoxides were realized with the sodium counter-ion, whereas the more reactive primary alkoxides required the harder, more Lewis acidic lithium counter-ion.

c-Myc is a bHLH-ZIP transcription factor that is responsible for the transcription of a wide range of target genes involved in many cancer-related cellular processes, such as proliferation, differentiation, apoptosis and metabolism. Over-expression of c-Myc has been observed in, and directly contributes to, a variety of human cancers including those of the hematopoietic system, lung, prostate and colon. To become transcriptionally active, c-Myc must first dimerize with Max via its own bHLH-ZIP domain. A proven strategy towards the inhibition of c-Myc oncogenic activity is to interfere with the structural integrity of the c-Myc–Max heterodimer. The small-molecule 10074-G5 is an inhibitor of c-Myc–Max dimerization (IC50 = 146 μM) that operates by binding and stabilizing c-Myc in its monomeric form. Herein, we report on our on-going efforts to optimize the c-Myc–Max inhibitory activity of 10074-G5-related molecules in vitro and in cancer cells that over-express c-Myc. Specifically, we have identified a congener of 10074-G5, termed 3jc48-3, that is about five times as potent (IC50 = 34 μM) at inhibiting c-Myc–Max dimerization as the parent compound. In addition, 3jc48-3 exhibited an approximate two-fold selectivity for c-Myc–Max heterodimers over Max–Max dimers, suggesting that, like its predecessor, its mode of action is through binding c-Myc. 3jc48-3 inhibited the proliferation of c-Myc-over-expressing HL60 and Daudi cells with single-digit micromolar IC50 values by causing growth arrest at the G0/G1 phase. Furthermore, co-immunoprecipitation studies indicated that 3jc48-3 inhibits c-Myc–Max dimerization in cells, which was further substantiated by the specific silencing of a c-Myc-driven luciferase reporter gene. Finally, unlike previously described 10074-G5 analogues, which are rapidly released and/or metabolized by cells following their uptake, 3jc48-3’s intracellular half-life was >17 h. Collectively, these data demonstrate 3jc48-3 to be one of the most potent cellularly active c-Myc inhibitors reported to date.

In order to mimic amphipathic α-helices, a novel scaffold based on a 1,2-diphenylacetylene was designed. NMR and computational modeling confirmed that an intramolecular hydrogen bond favors conformations of the 1,2-diphenylacetylene that allow for accurate mimicry of the i, i + 7 and i + 2, i + 5 side chains found on opposing faces of an α-helix.

We herein report on the pharmacophore determination of the ERK docking domain inhibitor (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione, which has led to the discovery of compounds with greater selectivities for inhibiting the proliferation of melanoma cells containing active ERK signaling.

A structure–activity relationship (SAR) study of the c-Myc (Myc) inhibitor 10074-G5 (N-([1,1′-biphenyl]-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine, 1) – which targets a hydrophobic domain of the Myc oncoprotein that is flanked by arginine residues – was executed in order to determine its pharmacophore. Whilst the 7-nitrobenzofurazan was found to be critical for inhibitory activity, the ortho-biphenyl could be replaced with a para-carboxyphenyl group to furnish the new inhibitor JY-3-094 (3q). Around five times as potent as the lead with an IC50 of 33 μM for disruption of the Myc–Max heterodimer, JY-3-094 demonstrated excellent selectivity over Max–Max homodimers, with no apparent effect at 100 μM. Importantly, the carboxylic acid of JY-3-094 improves the physicochemical properties of the lead compound, which will facilitate the incorporation of additional hydrophobicity that might enhance Myc inhibitory activity further still.

By conducting a structure-activity relationship study of the backbone of a series of oligoamide-foldamer-based α-helix mimetics of the Bak BH3 helix, we have identified especially potent inhibitors of Bcl-x(L). The most potent compound has a K(i) value of 94 nM in vitro, and single-digit micromolar IC(50) values against the proliferation of several Bcl-x(L)-overexpressing cancer cell lines.

The Ras→Raf→MEK(mitogen-activated kinase kinase)→ERK (extracellular-signal-regulated kinase) signalling pathway is one of at least five mitogen-activated protein kinase (MAPK) pathways that control several fundamental cellular processes, driving proliferation, differentiation and cell survival.[1–3] Signal transduction through this particular pathway, which is depicted in Figure 1, is initiated by the binding of a wide variety of ligands, including hormones and growth factors, to receptor tyrosine kinases (RTKs). This leads to the activation of Ras proteins (H-, K- and N-Ras isoforms) previously anchored in the plasma membrane by earlier post-translational reactions, e.g. farnesylation. Subsequently, the Ras proteins are induced to exchange their bound GDP for GTP, which leads to a conformational change in Ras and the initiation of a three-stage phosphorylation cascade that climaxes with the activation of ERK1/2. First, the Raf family of kinases (A-, B- and Raf-1 isoforms), the best studied of which is Raf-1, is recruited to the plasma membrane. Upon its subsequent phosphorylation, Raf-1 then activates (phosphorylates) MAP / ERK kinase 1 and 2 (MEK1/2), which, in turn, activate (phosphorylate) ERK1 and ERK2 (p44 MAPK and p42 MAPK, respectively). ERK1/2 are activated through phosphorylation of both a threonine and a tyrosine residue, namely Thr202 and Tyr204 of ERK1 and Thr183 and Tyr185 of ERK2. MEK1/2 are the only known activators of ERK1/2 and are, thus, dual specificity kinases. Activated ERK1/2 then phosphorylate serine/threonine residues of more than 50 downstream cytosolic and nuclear substrates, leading to alterations in gene expression profiles and an increase in proliferation, differentiation and cell survival.[1–3] Figure 1 Schematic representation of the Ras→Raf→MEK1/2→ERK1/2 signalling pathway. GF = growth factor, RTK = receptor tyrosine kinase, Grb2 = growth factor receptor-bound protein 2; Sos = son of sevenless; P indicates a phosphorylated serine, ... There is now considerable evidence that links the dysregulation of the Ras→Raf→MEK→ERK pathway to the oncogenesis of human cancers. Ras is hyperactivated in around 30% of human cancers, most commonly the K-Ras isoform.[4] More specifically, Ras activating mutations have been reported in about 90% of pancreatic carcinomas, 50% of colon carcinomas, 30% of lung cancers and in around 30% of myeloid leukaemia cases.[4] Activating mutations of Raf have also been reported in around 7% of human cancers.[5,6] In particular, mutations of B-Raf have been observed in over 60% of melanomas, around 30% of ovarian cancer and in approximately 20% of colorectal carcinomas, as well as in several other malignancies at lower frequencies.[5,6] Constitutively activate MEK1/2 and ERK1/2 proteins are present in a relatively high number of human tumours, particularly those from the colon, lung, pancreas, ovary and kidney.[7] Since mutations of the MEK1/2 and ERK1/2 genes have not been observed in human tumours, it seems probable that the hyperactivity of these proteins is a consequence of their constitutive phosphorylation due to hyperactivation of upstream effectors, including receptors, Ras and B-Raf. In summary, the Ras→Raf→MEK1/2→ERK1/2 pathway is an appealing target for the development of potential anti-cancer therapeutics. Moreover, the pathway offers several junctures for signal transduction blockade; due to the converging functions of MEK1/2 and ERK1/2, specific inhibition of these proteins is particularly desirable. In this mini-review, some of the more prominent small molecule inhibitors of the ERK pathway will be presented, with a particular emphasis on those discovered within the last ten to fifteen years. In the first section, we shall discuss those inhibitors that target proteins upstream of ERK1/2, specifically Raf and MEK1/2. We will then shift to the main focus of this review, which is the direct inhibition of ERK1/2 through targeting either the ATP-binding site (ATP-competitive inhibitors) or the surface of ERK and blocking its protein–protein interactions with its substrates (non-ATP-competitive inhibitors).

Transcription factors are DNA-binding proteins that – usually in combination with other proteins to form the pre-initiation complex (PIC) – regulate the transcription of specific DNA sequences (genes) into mRNA by controlling the recruitment of RNA polymerase II. Constitutive activation of transcription factors can lead to a variety of cancers, and are, therefore, important therapeutic targets. However, in stark contrast to targeting enzyme active sites, disruption of protein–protein or protein–DNA interactions involved in the transcriptional machinery is particularly challenging owing to the large interfacial areas involved, a lack of obvious binding sites and often non-contiguous contact points. Especially problematic for the development of small-molecules is the need by such agents to overcome the large free energy of association between protein–protein and, to a lesser extent, protein–DNA interfaces. Nevertheless, recent years have seen considerable success in this area of medicinal chemistry, cementing the notion that disruption of such interactions is feasible with small-molecule, drug-like compounds. We discuss, in particular, the disruption of dimeric transcription factors, such as STAT3–STAT3, c-Myc–Max and c-Jun–c-Fos (AP-1), with small-molecules that block their protein–protein interactions or their interactions with DNA.