Jeremy L. Yap

The novel BH3 α-helix mimetic JY-1-106 induces apoptosis in a subset of cancer cells (lung cancer, colon cancer and mesothelioma) by disrupting Bcl-xL and Mcl-1 protein-protein interactions with Bak.

BackgroundIt has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-2/Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. We designed the BH3 α-helix mimetic JY-1-106 to engage the hydrophobic BH3-binding grooves on the surfaces of both Bcl-xL and Mcl-1.MethodsJY-1-106–protein complexes were studied using molecular dynamics (MD) simulations and the SILCS methodology. We have evaluated the in vitro effects of JY-1-106 by using a fluorescence polarization (FP) assay, an XTT assay, apoptosis assays, and immunoprecipitation and western-blot assays. A preclinical human cancer xenograft model was used to test the efficacy of JY-1-106 in vivo.ResultsMD and SILCS simulations of the JY-1-106–protein complexes indicated the importance of the aliphatic side chains of JY-1-106 to binding and successfully predicted the improved affinity of the ligand for Bcl-xL over Mcl-1. Ligand binding affinities were measured via an FP assay using a fluorescently labeled Bak-BH3 peptide in vitro. Apoptosis induction via JY-1-106 was evidenced by TUNEL assay and PARP cleavage as well as by Bax–Bax dimerization. Release of multi-domain Bak from its inhibitory binding to Bcl-2/Bcl-xL and Mcl-1 using JY-1-106 was detected via immunoprecipitation (IP) western blotting.At the cellular level, we compared the growth proliferation IC50s of JY-1-106 and ABT-737 in multiple cancer cell lines with various Bcl-xL and Mcl-1 expression levels. JY-1-106 effectively induced cell death regardless of the Mcl-1 expression level in ABT-737 resistant solid tumor cells, whilst toxicity toward normal human endothelial cells was limited. Furthermore, synergistic effects were observed in A549 cells using a combination of JY-1-106 and multiple chemotherapeutic agents. We also observed that JY-1-106 was a very effective agent in inducing apoptosis in metabolically stressed tumors. Finally, JY-1-106 was evaluated in a tumor-bearing nude mouse model, and was found to effectively repress tumor growth. Strong TUNEL signals in the tumor cells demonstrated the effectiveness of JY-1-106 in this animal model. No significant side effects were observed in mouse organs after multiple injections.ConclusionsTaken together, these observations demonstrate that JY-1-106 is an effective pan-Bcl-2 inhibitor with very promising clinical potential.

Relaxation of the rigid backbone of an oligoamide-foldamer-based α-helix mimetic: identification of potent Bcl-xL inhibitors

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.

Small-Molecule Inhibitors of the ERK Signaling Pathway: Towards Novel Anticancer Therapeutics

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).

The downregulation of Mcl-1 via USP9X inhibition sensitizes solid tumors to Bcl-xl inhibition

BackgroundIt has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination.MethodsThe Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay.ResultsIn our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells.ConclusionMcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors.

Amphipathic α-helix mimetics based on a 1,2-diphenylacetylene scaffold.

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.

Small-molecule inhibitors of dimeric transcription factors: Antagonism of protein–protein and protein–DNA interactions

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.

Structural modifications of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione that improve selectivity for inhibiting the proliferation of melanoma cells containing active ERK signaling

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.

Structure-based design of N-substituted 1-hydroxy-4-sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncoprotein.

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 pocket 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 in the p2 pocket dominated affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 19-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 leukemia.

Structural Re-engineering of the α-Helix Mimetic JY-1-106 into Small Molecules: Disruption of the Mcl-1-Bak-BH3 Protein-Protein Interaction with 2,6-Di-Substituted Nicotinates.

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 1H–15N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts.

Discovery of methyl 4'-methyl-5-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-[1,1'-biphenyl]-3-carboxylate, an improved small-molecule inhibitor of c-Myc-max dimerization.

c‐Myc is a basic helix‐loop‐helix‐leucine zipper (bHLH‐ZIP) transcription factor that is responsible for the transcription of a wide range of target genes involved in many cancer‐related cellular processes. 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 Myc‐associated factor X (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. We have identified a congener of 10074‐G5, termed 3jc48‐3 (methyl 4′‐methyl‐5‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)‐[1,1′‐biphenyl]‐3‐carboxylate), that is about five times as potent (IC50=34 μM) at inhibiting c‐Myc–Max dimerization as the parent compound. 3jc48‐3 exhibited an approximate twofold selectivity for c‐Myc–Max heterodimers over Max–Max homodimers, suggesting that 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. 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, 3jc48‐3′s intracellular half‐life was >17 h. Collectively, these data demonstrate 3jc48‐3 to be one of the most potent, cellularly active and stable c‐Myc inhibitors reported to date.