ABSTRACT
We report the identification of KD5170, a potent mercaptoketone-based Class I and II-histone deacetylase inhibitor that demonstrates broad spectrum cytotoxic activity against a range of human tumor-derived cell lines. KD5170 exhibits robust and sustained histone H3 hyperacetylation in HCT-116 xenograft tumors following single oral or i.v. dose and inhibition of tumor growth following chronic dosing.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Prodrugs/pharmacology , Pyridines/pharmacology , Sulfonamides/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Drug Screening Assays, Antitumor , HCT116 Cells , Humans , Inhibitory Concentration 50 , Mice , Mice, Nude , Molecular Structure , Prodrugs/chemistry , Pyridines/chemistry , Structure-Activity Relationship , Sulfonamides/chemistry , Xenograft Model Antitumor AssaysABSTRACT
In an effort to discover novel non-hydroxamic acid histone deacetylase (HDAC) inhibitors, a novel alpha-mercaptoketone was identified in a high-throughput screen. Lead optimization of the screening hit, led to a number of potent HDAC inhibitors. In particular, alpha-mercaptoketone 19y (KD5150) exhibited nanomolar in vitro activity and inhibition of tumor growth in vivo.
Subject(s)
Drug Screening Assays, Antitumor , Histone Deacetylase Inhibitors , Ketones/chemistry , Antineoplastic Agents/therapeutic use , Chelating Agents/pharmacology , Chemistry, Pharmaceutical , Drug Design , Enzyme Inhibitors/pharmacology , HeLa Cells , Humans , Models, Chemical , Neoplasms/drug therapy , Prodrugs/chemistry , Structure-Activity Relationship , Zinc/chemistryABSTRACT
Histone deacetylase (HDAC) inhibitors have garnered significant attention as cancer drugs. These therapeutic agents have recently been clinically validated with the market approval of vorinostat (SAHA, Zolinza) for treatment of cutaneous T-cell lymphoma. Like vorinostat, most of the small-molecule HDAC inhibitors in clinical development are hydroxamic acids, whose inhibitory activity stems from their ability to coordinate the catalytic Zn2+ in the active site of HDACs. We sought to identify novel, nonhydroxamate-based HDAC inhibitors with potentially distinct pharmaceutical properties via an ultra-high throughput small molecule biochemical screen against the HDAC activity in a HeLa cell nuclear extract. An alpha-mercaptoketone series was identified and chemically optimized. The lead compound, KD5170, exhibits HDAC inhibitory activity with an IC50 of 0.045 micromol/L in the screening biochemical assay and an EC50 of 0.025 micromol/L in HeLa cell-based assays that monitor histone H3 acetylation. KD5170 also exhibits broad spectrum classes I and II HDAC inhibition in assays using purified recombinant human isoforms. KD5170 shows significant antiproliferative activity against a variety of human tumor cell lines, including the NCI-60 panel. Significant tumor growth inhibition was observed after p.o. dosing in human HCT-116 (colorectal cancer), NCI-H460 (non-small cell lung carcinoma), and PC-3 (prostate cancer) s.c. xenografts in nude mice. In addition, a significant increase in antitumor activity and time to end-point occurred when KD5170 was combined with docetaxel in xenografts of the PC-3 prostate cancer cell line. The biological and pharmaceutical profile of KD5170 supports its continued preclinical and clinical development as a broad spectrum anticancer agent.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Pyridines/pharmacology , Sulfonamides/pharmacology , Animals , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Drug Screening Assays, Antitumor , Female , Humans , Inhibitory Concentration 50 , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Prostatic Neoplasms/drug therapy , Xenograft Model Antitumor AssaysABSTRACT
We have identified and synthesized a series of thiophene containing inhibitors of kinesin spindle protein. SAR studies led to the synthesis of 33, which was co-crystallized with KSP and determined to bind to an allosteric pocket previously described for other known KSP inhibitors.
Subject(s)
Chemistry, Pharmaceutical/methods , Kinesins/antagonists & inhibitors , Thiophenes/chemistry , Allosteric Site , Amides/chemistry , Antimitotic Agents/pharmacology , Cell Line, Tumor , Crystallization , Crystallography, X-Ray , Drug Design , Humans , Inhibitory Concentration 50 , Kinesins/chemistry , Models, Chemical , Models, Molecular , Molecular ConformationSubject(s)
Adenosine Triphosphate/analogs & derivatives , Antibodies/chemistry , DNA Probes/chemistry , DNA/chemistry , Fluorescent Dyes/chemistry , Stilbenes/chemistry , Adenosine Triphosphate/chemical synthesis , Adenosine Triphosphate/chemistry , DNA Probes/chemical synthesis , Fluorescent Dyes/chemical synthesis , Molecular StructureABSTRACT
Despite the enormous health risks, people continue to smoke and use tobacco primarily as a result of nicotine addiction. As part of our immunopharmacotherapy research, the effects of active and passive immunizations on acute nicotine-induced locomotor activity in rats were investigated. To this end, rats were immunized with either a NIC-KLH immunoconjugate vaccine designed to elicit an antinicotine immune response, or were administered an antinicotine monoclonal antibody, NIC9D9, prior to a series of nicotine challenges and testing sessions. Vaccinated rats showed a 45% decrease in locomotor activity compared to a 16% decrease in controls. Passive immunization with NIC9D9 resulted in a 66.9% decrease in locomotor activity versus a 3.4% decrease in controls. Consistent with the behavioral data, much less nicotine was found in the brains of immunized rats. The results support the potential clinical value of immunopharmacotherapy for nicotine addiction in the context of tobacco cessation programs.
Subject(s)
Immunization , Motor Activity , Nicotine/antagonists & inhibitors , Nicotine/immunology , Animals , Brain/metabolism , Hemocyanins/chemistry , Male , Molecular Structure , Motor Activity/drug effects , Nicotine/analysis , Nicotine/pharmacology , Rats , Rats, WistarABSTRACT
A conformationally constrained hapten is presented that is capable of catalyzing the first antibody-mediated photo-Fries rearrangement. In this reaction, absorption of light energy by a diphenyl ether substrate results in homolytic C-O bond cleavage followed by recombination to yield biphenyl-derived products. The most proficient antibody studied converts 4-phenoxyaniline 15 into 2-hydroxy-5-aminobiphenyl 16 under high-intensity irradiation at a rate of 8.6 microM/min. These results support a recent hypothesis stating that immunization with conformationally constrained haptens provides higher titers for the acquisition of simple binding antibodies; however, in this case, conformational constraint does not ensure the development of more efficient catalysts. Using the obtained antibodies, the presence of products resulting from escape of free radicals from the solvent cage can be suppressed, altering the excited state energy surface such that free radicals are funneled into the formation of the desired biphenyl product. However, studies also show the inactivation of the antibodies as a result of photodecay of the biphenyl product. Using an isocyanate scavenging resin, the photodecay product could be removed and the inactivation of the antibody drastically reduced. Furthermore, despite the observed photodecay, turnover of the antibody was present; this represents the first case in which true turnover of a photochemical reaction using a catalytic antibody could be observed.
Subject(s)
Aminobiphenyl Compounds/chemistry , Aniline Compounds/chemistry , Antibodies/chemistry , Haptens/chemistry , Phenyl Ethers/chemistry , Aminobiphenyl Compounds/chemical synthesis , Catalysis , Photochemistry , ThermodynamicsABSTRACT
Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.
Subject(s)
Breast Neoplasms/immunology , Immunoglobulin Fragments/immunology , Immunoglobulin Gm Allotypes/immunology , Melanoma/immunology , Amino Acid Sequence , Antibody Specificity , Carbohydrate Sequence , Flow Cytometry , Humans , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/genetics , Immunoglobulin Gm Allotypes/chemistry , Kinetics , Molecular Sequence Data , Peptide Library , Thermodynamics , Tumor Cells, CulturedABSTRACT
One aspect of immunopharmacotherapy for cocaine abuse involves the use of a catalytic monoclonal antibody (mAb) to degrade cocaine via hydrolysis of the benzoate ester. A cocaine benzoylthio ester analogue provides a means to implement high-throughput selection strategies to potentially isolate mAbs with high activity. The required analogue was synthesized starting from (-)-cocaine hydrochloride and possessed the cocaine absolute configuration. Key points in the preparation were the introduction of the sulfur atom at C-3 via a bromomagnesium thiolate addition to the exo face of anhydroecgonine, separation of C-2 diastereomers, recycling of a C-2 thio ester byproduct, and formation of the necessary C-2 methyl and C-3 benzoylthio esters. Effects resulting from the lower electronegativity and greater hydrophobicity of sulfur compared to oxygen were observed. These characteristics could result in interesting drug properties. Furthermore, the analogue was found to be a substrate for catalytic mAbs that hydrolyze cocaine as monitored by HPLC and also spectrophotometry by coupling cleavage of the benzoylthio ester to the disulfide exchange with Ellman's reagent. Screening antibody libraries with the new cocaine analogue using the spectroscopic assay provides an avenue for the high-throughput identification of catalysts that efficiently breakdown cocaine.
