Probing p300/CBP associated Factor (PCAF)-dependent pathways with a small molecule inhibitor

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.

Vasoactivity of AG014699, a clinically active small molecule inhibitor of poly(ADP-ribose) polymerase: a contributory factor to chemopotentiation in vivo?

Purpose: Poly(ADP-ribose) polymerase (PARP) plays an important role in DNA repair, and PARP inhibitors can enhance the activity of DNA-damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in phase II clinical development. However, the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitization, which could widen its clinical application.

Experimental Design: Temozolomide response was analyzed in vitro and in vivo. Vessel dynamics were monitored using “mismatch” following the administration of perfusion markers and real-time analysis of fluorescently labeled albumin uptake in to tumors established in dorsal window chambers. Further mechanistic investigations used ex vivo assays of vascular smooth muscle relaxation, gut motility, and myosin light chain kinase (MLCK) inhibition.

Results: AG014699 failed to sensitize SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1 mg/kg) improved tumor perfusion comparably with the control agents nicotinamide (1 g/kg) and AG14361 (forerunner to AG014699; 10 mg/kg). AG014699 and AG14361 relaxed preconstricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited MLCK at concentrations that relaxed isolated arteries, whereas AG14361 had no effect.

Conclusion: Increased vessel perfusion elicited by AG014699 could increase tumor drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefit.

A novel small-molecule inhibitor of IL-6 signalling.

A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.

Differential inhibition sensitivities of MET mutants to the small molecule inhibitor SU11274

Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC(50) values for autophosphorylation inhibition ranging between 0.15 and 1.5muM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCgamma and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain.

Cell cycle reactivation of cochlear progenitor cells in neonatal FUCCI mice by a GSK3 small molecule inhibitor.

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

CHARACTERIZATION OF SELECTIVE INHIBITION OF ADENYLYL CYCLASE ACTIVITY BY SMALL MOLECULE INHIBITOR NKY80

The nine membrane-bound isoforms of adenylyl cyclase (AC), via synthesis of the signaling molecule cyclic AMP (cAMP), are involved in many isoform specific physiological functions. Decreasing AC5 activity has been shown to have potential therapeutic benefit, including reduced stress on the heart, pain relief, and attenuation of morphine dependence and withdrawal behaviors. However, AC structure is well conserved, and there are currently no isoform selective AC inhibitors in clinical use. P-site inhibitors inhibit AC directly at the catalytic site, but with an uncompetitive or noncompetitive mechanism. Due to this mechanism and nanomolar potency in cell-free systems, attempts at ligand-based drug design of novel AC inhibitors frequently use P-site inhibitors as a starting template. One small molecule inhibitor designed through this process, NKY80, is described as an AC5 selective inhibitor with low micromolar potency in vitro. P-site inhibitors reveal important ligand binding “pockets” in the AC catalytic site, but specific interactions that give NKY80 selectivity are unclear. Identifying and characterizing unique interactions between NKY80 and AC isoforms would significantly aid the development of isoform selective AC inhibitors. I hypothesized that NKY80’s selective inhibition is conferred by AC isoform specific interactions with the compound within the catalytic site. A structure-based virtual screen of the AC catalytic site was used to identify novel small molecule AC inhibitors. Identified novel inhibitors are isoform selective, supporting the catalytic site as a region capable of more potent isoform selective inhibition. Although NKY80 is touted commercially as an AC5 selective inhibitor, its characterization suggests strong inhibition of both AC5 and the closely related AC6. NKY80 was also virtually docked to AC to determine how NKY80 binds to the catalytic site. My results show a difference between NKY80 binding and the conformation of classic P-site inhibitors. The selectivity and notable differences in NKY80 binding to the AC catalytic site suggest a catalytic subregion more flexible in AC5 and AC6 that can be targeted by selective small molecule inhibitors.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

HIV-1 entry into CD4+ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

Rapid synthesis and zebrafish evaluation of a phenanthridine-based small molecule library

A Heck cyclisation approach is described for the rapid synthesis of a library of natural product-like small molecules, based on the phenanthridine core. The synthesis of a range of substituted benzylamine building blocks and their incorporation into the library is reported, together with a highly selective cis-dihydroxylation protocol that enables access to the target compounds in an efficient manner. Biological evaluation of the library using zebrafish phenotyping has led to the discovery of compound 20c, a novel inhibitor of early-stage zebrafish embryo development.

Flat SAR of P3-methylsulphonamide based small molecule legumain inhibitors

This letter describes the design, development and SAR exploration of a novel series of small legumain inhibitors. The SAR of a new small molecule legumain inhibitor chemotype was explored and found to have improved physiochemical properties compared to previously developed inhibitors within our group. However, further development of this series was found to be limited as the SAR was observed to be relatively flat.

Targeting the p53–MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

The human p53 tumor suppressor, known as the “guardian of the genome”, is one of the most important molecules in human cancers. One mechanism for suppressing p53 uses its negative regulator, MDM2, which modulates p53 by binding directly to and decreasing p53 stability. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-Acute lymphoblastic leukemia (ALL) patient samples. In this study we demonstrated that treatment with Nutlin-3a induced grow arrest and apoptosis mediated by p53 pathway in ALL cells with wild-type p53, in time and dose-dependent manner. Consequently, MDM2 inhibitor caused an increase of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from Ph+ ALL patients with the T315I Bcr-Abl kinase domain mutation. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis in sensitive cell lines was performed. A total of 621 genes were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change -1.35 and -1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21. Given the importance of BMI in the control of apoptosis, we investigated its pattern in treated and untreated cells, confirming a marked decrease after exposure to MDM2 inhibitor in ALL cells. Noteworthy, the BMI-1 levels remained constant in resistant cells. Therefore, BMI-1 may be used as a biomarker of response. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph-ALL.

Identification and Characterization of New Small Molecule Inhibitors of Picornavirus Replication

The Picornaviridae family consists of positive-strand RNA viruses that are the causative agents of a variety of diseases in humans and animals. Few drugs targeting picornaviruses are available, making the discovery of new antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. The antiviral effect of these compounds is not likely related to their known cellular targets because other inhibitors targeting the same pathways did not inhibit viral replication. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited the formation of a functional replication complex, while E5(1) and E7(2) affected replication after the replication complex had formed. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Poliovirus resistant to E7(2) had a single mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target either PI4KIIIβ (major enviroxime-like compounds) or OSBP (minor enviroxime-like compounds), cellular factors involved in lipid metabolism and shown to be important for replication of diverse positive-strand RNA viruses. We classified E7(2) as a minor enviroxime-like compound, because the localization of OSBP changed in the presence of this inhibitor. Interestingly, both E7(2) and major enviroxime-like compound GW5074 interfered with the viral polyprotein processing. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. Studies with these compounds shed light on pathways shared by diverse picornaviruses that could be potential targets for the development of broad-spectrum antiviral drugs.

The synthesis of novel nitroxides and their application as small-molecule antioxidants and profluorescent probes for oxidative stress

The detection and potential treatment of oxidative stress in biological systems has been explored using isoindoline-based nitroxide radicals. A novel tetraethyl-fluorescein nitroxide was synthesised for its use as a profluorescent probe for redox processes in biological systems. This tetraethyl system, as well as a tetramethyl-fluorescein nitroxide, were shown to be sensitive and selective probes for superoxide in vitro. The redox environment of cellular systems was also explored using the tetramethylfluorescein species based on its reduction to the hydroxylamine. Flow cytometry was employed to assess the extent of nitroxide reduction, reflecting the overall cellular redox environment. Treatment of normal fibroblasts with rotenone and 2-deoxyglucose resulted in an oxidising cellular environment as shown by the lack of reduction of the fluorescein-nitroxide system. Assessment of the tetraethyl-fluorescein nitroxide system in the same way demonstrated its enhanced resistance to reduction and offers the potential to detect and image biologically relevant reactive oxygen species directly. Importantly, these profluorescent nitroxide compounds were shown to be more effective than the more widely used and commercially available probes for reactive oxygen species such as 2’,7’-dichlorodihydrofluorescein diacetate. Fluorescence imaging of the tetramethyl-fluorescein nitroxide and a number of other rhodamine-nitroxide derivatives was undertaken, revealing the differential cellular localisation of these systems and thus their potential for the detection of redox changes in specific cellular compartments. As well as developing novel methods for the detection of oxidative stress, a number of novel isoindoline nitroxides were synthesised for their potential application as small-molecule antioxidants. These compounds incorporated known pharmacophores into the isoindoline-nitroxide structure in an attempt to increase their efficacy in biological systems. A primary and a secondary amine nitroxide were synthesised which incorporated the phenethylamine backbone of the sympathomimetic amine class of drugs. Initial assessment of the novel primary amine derivative indicated a protective effect comparable to that of 5-carboxy-1,1,3,3- tetramethylisoindolin-2-yloxyl. Methoxy-substituted nitroxides were also synthesised as potential antioxidants for their structural similarity to some amphetamine type stimulants. A copper-catalysed methodology provided access to both the mono- and di-substituted methoxy-nitroxides. Deprotection of the ethers in these compounds using boron tribromide successfully produced a phenolnitroxide, however the catechol moiety in the disubstituted derivative appeared to undergo reaction with the nitroxide to produce quinone-like degradation products. A novel fluoran-nitroxide was also synthesised from the methoxy-substituted nitroxide, providing a pH-sensitive spin probe. An amino-acid precursor containing a nitroxide moiety was also synthesised for its application as a dual-action antioxidant. N-Acetyl protection of the nitroxide radical was necessary prior to the Erlenmeyer reaction with N-acetyl glycine. Hydrolysis and reduction of the azlactone intermediate produced a novel amino acid precursor with significant potential as an effective antioxidant.