Drugging "undruggable" genes for cancer treatment: Are we making progress?

RAS, TP53 (p53) and MYC are among the most frequently altered driver genes in cancer. Thus, RAS is the most frequently mutated oncogene, MYC the most frequently amplified gene and TP53 the most frequently mutated tumor suppressor gene and overall the most frequently mutated gene in cancer. Theoretically, therefore, these genes are highly attractive targets for cancer treatment. However, as the protein products of each of these genes lack an accessible hydrophobic pocket into which low molecular weight compounds might bind with high affinity, they have proved difficult to target and have traditionally been referred to as "undruggable." Despite this branding, several low molecular weight compounds targeting each of these proteins have recently been reported to have anticancer activity in preclinical models. Indeed, several drugs inhibiting mutant KRAS, MYC overexpression or reactivating mutant p53 have undergone or are currently undergoing clinical trials. For targeting mutant KRAS and reactivating mutant p53, trials have progressed to a Phase III stage, that is, the mutant-p53 reactivating drug, APR-246 is currently being investigated in patients with myelodysplastic syndrome (MDS) and the RAS inhibitor, rigosertib is also undergoing evaluation in patients with MDS. Although there appears to be no directly acting MYC inhibitor currently being tested in a clinical trial, an anti-MYC compound, known as OmoMYC has been extensively validated in multiple preclinical models and is being developed for clinical evaluation. Based on current evidence, the traditional perception of RAS, p53 and MYC as being "undruggable" would appear to be coming to an end.

Computational study on natural compounds inhibitor of c-Myc.

To screen and identify ideal leading compounds from a drug library (ZINC15 database) with potential inhibition effect against c-Myc to contribute to medication design and development.A series of computer-aided virtual screening techniques were performed to identify potential inhibitors of c-Myc. LibDock from the software Discovery Studio was used to do a structure-based screening after ADME (absorption, distribution, metabolism, excretion) and toxicity prediction. Molecular docking was utilized to show the binding affinity and potential mechanism between ligands and c-Myc. Stability of the ligand-receptor complex was analyzed by molecular dynamic simulation at the end of the research.Compounds with more interactive energy which are confirmed to be the potential inhibitors for c-Myc were identified from the ZINC15 databases. Additionally, those compounds are also anticipated with fewer ames mutagenicity, rodent carcinogenicity, nondevelopmental toxic potential, and tolerant with cytochrome p450 2D6(CYP2D6). Dynamic simulation analysis also revealed that the very compounds had more favorable potential energy compared with 10058-F4(ZINC12406714). Furthermore, we prove that those compounds are stable and can exist in natural conditions.This study demonstrates that the compounds are potential therapeutic inhibitors for c-Myc. These compounds are safe and stable for drug candidates and may play a critical role in c-Myc inhibitor development.

Network pharmacology based virtual screening of active constituents of Prunella vulgaris L. and the molecular mechanism against breast cancer.

Prunella vulgaris L, a perennial herb widely used in Asia in the treatment of various diseases including cancer. In vitro studies have demonstrated the therapeutic effect of Prunella vulgaris L. against breast cancer through multiple pathways. However, the nature of the biological mechanisms remains unclear. In this study, a Network pharmacology based approach was used to explore active constituents and potential molecular mechanisms of Prunella vulgaris L. for the treatment of breast cancer. The methods adopted included active constituents prescreening, target prediction, GO and KEGG pathway enrichment analysis. Molecular docking experiments were used to further validate network pharmacology results. The predicted results showed that there were 19 active ingredients in Prunella vulgaris L. and 31 potential gene targets including AKT1, EGFR, MYC, and VEGFA. Further, analysis of the potential biological mechanisms of Prunella vulgaris L. against breast cancer was performed by investigating the relationship between the active constituents, target genes and pathways. Network analysis showed that Prunella vulgaris L. exerted a promising preventive effect on breast cancer by acting on tumor-associated signaling pathways. This provides a basis to understand the mechanism of the anti-breast cancer activity of Prunella vulgaris L.

Structural and Biophysical Insights into the Function of the Intrinsically Disordered Myc Oncoprotein.

Myc is a transcription factor driving growth and proliferation of cells and involved in the majority of human tumors. Despite a huge body of literature on this critical oncogene, our understanding of the exact molecular determinants and mechanisms that underlie its function is still surprisingly limited. Indubitably though, its crucial and non-redundant role in cancer biology makes it an attractive target. However, achieving successful clinical Myc inhibition has proven challenging so far, as this nuclear protein is an intrinsically disordered polypeptide devoid of any classical ligand binding pockets. Indeed, Myc only adopts a (partially) folded structure in some contexts and upon interacting with some protein partners, for instance when dimerizing with MAX to bind DNA. Here, we review the cumulative knowledge on Myc structure and biophysics and discuss the implications for its biological function and the development of improved Myc inhibitors. We focus this biophysical walkthrough mainly on the basic region helix-loop-helix leucine zipper motif (bHLHLZ), as it has been the principal target for inhibitory approaches so far.

Structure-based Inhibitor Design for the Intrinsically Disordered Protein c-Myc.

Intrinsically disordered proteins (IDPs) are associated with various diseases and have been proposed as promising drug targets. However, conventional structure-based approaches cannot be applied directly to IDPs, due to their lack of ordered structures. Here, we describe a novel computational approach to virtually screen for compounds that can simultaneously bind to different IDP conformations. The test system used c-Myc, an oncoprotein containing a disordered basic helix-loop-helix-leucine zipper (bHLH-LZ) domain that adopts a helical conformation upon binding to Myc-associated factor X (Max). For the virtual screen, we used three binding pockets in representative conformations of c-Myc370-409, which is part of the disordered bHLH-LZ domain. Seven compounds were found to directly bind c-Myc370-409 in vitro, and four inhibited the growth of the c-Myc-overexpressing cells by affecting cell cycle progression. Our approach of IDP conformation sampling, binding site identification, and virtual screening for compounds that can bind to multiple conformations provides a useful strategy for structure-based drug discovery targeting IDPs.

The pH sensibility of actin-bundling LIM proteins is governed by the acidic properties of their C-terminal domain.

Actin-bundling Arabidopsis LIM proteins are subdivided into two subfamilies differing in their pH sensitivity. Widely-expressed WLIMs are active under low and high physiologically-relevant pH conditions, whereas pollen-enriched PLIMs are inactivated by pH values above 6.8. By a domain swapping approach we identified the C-terminal (Ct) domain of PLIMs as the domain responsible for pH responsiveness. Remarkably, this domain conferred pH sensitivity to LIM proteins, when provided "in trans" (i.e., as a single, independent, peptide), indicating that it operates through the interaction with another domain. An acidic 6xc-Myc peptide functionally mimicked the Ct domain of PLIMs and efficiently inhibited LIM actin bundling activity under high pH conditions. Together, our data suggest a model where PLIMs are regulated by an intermolecular interaction between their acidic Ct domain and another, yet unidentified, domain.

Perturbation of the c-Myc-Max protein-protein interaction via synthetic α-helix mimetics.

The rational design of inhibitors of the bHLH-ZIP oncoprotein c-Myc is hampered by a lack of structure in its monomeric state. We describe herein the design of novel, low-molecular-weight, synthetic α-helix mimetics that recognize helical c-Myc in its transcriptionally active coiled-coil structure in association with its obligate bHLH-ZIP partner Max. These compounds perturb the heterodimer's binding to its canonical E-box DNA sequence without causing protein-protein dissociation, heralding a new mechanistic class of "direct" c-Myc inhibitors. In addition to electrophoretic mobility shift assays, this model was corroborated by further biophysical methods, including NMR spectroscopy and surface plasmon resonance. Several compounds demonstrated a 2-fold or greater selectivity for c-Myc-Max heterodimers over Max-Max homodimers with IC50 values as low as 5.6 µM. Finally, these compounds inhibited the proliferation of c-Myc-expressing cell lines in a concentration-dependent manner that correlated with the loss of expression of a c-Myc-dependent reporter plasmid despite the fact that c-Myc-Max heterodimers remained intact.

Inhibitor of MYC identified in a Kröhnke pyridine library.

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The Kd of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.

Coadministration of antigen-conjugated and free CpG: effects of in vitro and in vivo interactions in a murine model.

CpG oligodeoxynucleotides (CpG) are widely studied as promising adjuvants in vaccines against a range of diseases including infection, cancer or allergy. Conjugating antigen to CpG has been shown to potentiate the adjuvant effect via enhancing antigen uptake and danger signaling by the very same cell. In the present study, using biotinylated CpG and streptavidin as a model system, we demonstrate that CpG motif containing free and antigen-conjugated oligonucleotides do not compete in terms of cell activation via TLR9, but do compete for cellular uptake. Antigen-conjugated CpG enhances cellular association and uptake of the antigen by antigen-presenting cells (APC) and T cells. Free CpG efficiently competes with antigen-CpG conjugates in BMDC and T cells, but shows weak or no competition in B cells that have higher TLR9 expression. Vaccination with antigen-conjugated CpG or with a mixture of antigen and CpG elevates the level of antigen-specific antibodies but co-administration of CpG-antigen conjugates and free CpG adversely effects immunogenicity. These observations may help optimize CpG-based vaccine formulation.

Silencing c-MYC expression by targeting quadruplex in P1 promoter using locked nucleic acid trap.

The nuclease hypersensitive element of P1 promoter in c-MYC gene harbors a potential of unusual structure called quadruplex, which is involved in molecular recognition and function. This Hoogsteen bonded structure is in dynamic equilibrium with the usual Watson-Crick duplex structure, and these competing secondary structures undergo interconversion for execution of their respective biological roles. Herein, we investigate the sensitivity of the c-MYC quadruplex-duplex equilibrium by employing a locked nucleic acid (LNA) modified complementary strand as a pharmacological agent. Our biophysical experiments indicate that the c-MYC quadruplex under physiological conditions is stable and dominates the quadruplex-WC duplex equilibrium in both sodium and potassium buffers. This equilibrium is perturbed upon introducing the LNA modified complementary strand, which demonstrates efficient invasion of stable c-MYC quadruplex and duplex formation in contrast to the unmodified complementary strand. Our data indicate that LNA modifications confer increased thermodynamic stability to the duplex and thus favor the predominance of the duplex population over that of the quadruplex. Further, we demonstrate that this perturbation of equilibrium by a pharmacological agent results in altered gene expression. Our in vivo experiment performed using the LNA modified complementary strand suggests the influence of the quadruplex-duplex structural switch in the modulation of gene expression. We believe that this exploratory approach utilizing the selectivity and specificity of Watson-Crick base pairing of LNA bases would allow the modulation of quadruplex regulated gene expression.

Structure and function of the c-myc DNA-unwinding element-binding protein DUE-B.

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

Topology of NAT2, a prototypical example of a new family of amino acid transporters.

Amino acids are the predominant form of nitrogen available to the heterotrophic tissues of plants. These essential organic nutrients are transported across the plasma membrane of plant cells by proton-amino acid symporters. Our lab has cloned an amino acid transporter from Arabidopsis, NAT2/AAP1, that represents the first example of a new class of membrane transporters. We are investigating the structure and function of this porter because it is a member of a large gene family in plants and because its wide expression pattern suggests it plays a central role in resource allocation. In the results reported here, we investigated the topology of NAT2 by engineering a c-myc epitope on either the N or C terminus of the protein. We then used in vitro translation, partial digestion with proteinase K, and immunoprecipitation to identify a group of oriented peptide fragments. We modeled the topology of NAT2 based on the lengths of the peptide fragments that allowed us to estimate the location of protease accessible cleavage sites. We independently identified the location of the N and C termini using immunofluorescence microscopy of NAT2 expressed in COS-1 cells. We also investigated the glycosylation status of several sites of potential N-linked glycosylation. Based on the combined data, we propose a novel 11 transmembrane domain model with the N terminus in the cytoplasm and C terminus facing outside the cell. This model of protein topology anchors our complementary investigations of porter structure and function using site-directed and random mutagenesis.

The cysteine-rich protein family of highly related LIM domain proteins.

Here we describe a family of closely related LIM domain proteins in avian cells. The LIM motif defines a zinc-binding domain that is found in a variety of transcriptional regulators, proto-oncogene products, and proteins associated with sites of cell-substratum contact. One type of LIM-domain protein, called the cysteine-rich protein (CRP), is characterized by the presence of two LIM domains linked to short glycine-rich repeats and a potential nuclear localization signal. We have identified and characterized two evolutionarily conserved members of the CRP family, CRP1 and CRP2, in chicken and quail. Expression of the genes encoding both CRP1 and CRP2 is differentially regulated in normal versus transformed cells, raising the possibility that members of the CRP family may function in control of cell growth and differentiation.