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1.
ACS Med Chem Lett ; 14(12): 1631-1639, 2023 Dec 14.
Article in English | MEDLINE | ID: mdl-38116426

ABSTRACT

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

2.
J Pept Sci ; 20(1): 7-19, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24222478

ABSTRACT

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.


Subject(s)
Anti-Obesity Agents/chemical synthesis , Hypoglycemic Agents/chemical synthesis , Neuropeptides/chemical synthesis , Neuropeptides/pharmacology , Polyethylene Glycols/pharmacology , Serum Albumin/chemical synthesis , Animals , Anti-Obesity Agents/pharmacokinetics , Anti-Obesity Agents/pharmacology , Blood Glucose , Cell Line , Drug Evaluation, Preclinical , Humans , Hypoglycemic Agents/pharmacokinetics , Hypoglycemic Agents/pharmacology , Male , Mice , Mice, Inbred C57BL , Neuropeptides/pharmacokinetics , Polyethylene Glycols/pharmacokinetics , Receptors, Neurotransmitter/agonists , Serum Albumin/pharmacokinetics , Serum Albumin/pharmacology , Serum Albumin, Human , Weight Loss/drug effects
3.
Nucleic Acids Res ; 41(15): e145, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23766292

ABSTRACT

Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.


Subject(s)
Oligonucleotides/chemistry , Polymerase Chain Reaction/methods , RNA, Messenger/genetics , RNA, Untranslated/chemistry , Whole Body Imaging/methods , Animals , HCT116 Cells , Humans , Image Processing, Computer-Assisted , Male , Mice , Oligonucleotides/pharmacokinetics , Oligonucleotides/therapeutic use , Organ Specificity , RNA, Messenger/chemistry , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , RNA, Untranslated/genetics , Reproducibility of Results , Sensitivity and Specificity , Tissue Distribution
4.
PLoS One ; 3(1): e1508, 2008 Jan 30.
Article in English | MEDLINE | ID: mdl-18231595

ABSTRACT

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.


Subject(s)
Antibodies, Monoclonal/genetics , Bacteriophages/genetics , Oligonucleotide Array Sequence Analysis , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacokinetics , Enzyme-Linked Immunosorbent Assay , Immunoglobulin G/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Surface Plasmon Resonance
5.
J Med Chem ; 47(1): 14-7, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-14695815

ABSTRACT

alpha,gamma-Diketo acids (DKA) were discovered from screening as selective and reversible inhibitors of hepatitis C virus NS5b RNA-dependent RNA polymerase. The diketo acid moiety proved essential for activity, while substitution on the gamma position was necessary for selectivity and potency. Optimization led to the identification of a DKA inhibitor of NS5b polymerase with IC(50) = 45 nM, one of the most potent HCV NS5b polymerase inhibitors reported.


Subject(s)
Keto Acids/chemical synthesis , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Viral Nonstructural Proteins/physiology , Keto Acids/chemistry , Models, Molecular , RNA-Dependent RNA Polymerase/chemistry , Structure-Activity Relationship
6.
Bioconjug Chem ; 14(2): 276-81, 2003.
Article in English | MEDLINE | ID: mdl-12643736

ABSTRACT

Oligomers with two identical peptide nucleic acid sequences joined by a flexible hairpin linker (bisPNA) can stably bind to specific DNA sequences without altering plasmid supercoiling, thus offering a unique opportunity to attach various functional entities to high molecular weight DNA. Current synthetic approaches, however, severely limit the possibility to link peptides or other chemical moieties (i.e., sugars, oligonucleotides, etc.) to bisPNA. Here we report a novel strategy for the synthesis of bisPNA-peptide conjugates in which chemoselective ligation of bisPNA to peptides was accomplished through oxime formation between an oxy-amine-containing peptide and a bisPNA-methyl ketone (complementary modifications can also be used). The described synthesis is highly efficient, does not require a protection strategy, and is carried out under mild aqueous conditions. Through this methodology long peptide sequences in either C to N or N to C polarity can be linked to bisPNA. In addition, this protocol makes the conjugation of cysteine-containing peptides feasible and allows disulfide bond formation to be controlled. This same approach can be exploited to link oligonucleotides, sugars, or other chemical entities to bisPNA.


Subject(s)
Oximes/chemical synthesis , Peptide Nucleic Acids/chemical synthesis , Amino Acid Sequence , Chromatography, High Pressure Liquid , DNA/metabolism , Indicators and Reagents , Mass Spectrometry , Molecular Sequence Data , Oligonucleotide Probes/chemical synthesis , Oligonucleotide Probes/chemistry , Oximes/chemistry , Peptide Nucleic Acids/chemistry , Protein Binding , Recombinant Fusion Proteins/chemical synthesis , Recombinant Fusion Proteins/chemistry
7.
Bioconjug Chem ; 13(3): 676-8, 2002.
Article in English | MEDLINE | ID: mdl-12009961

ABSTRACT

The synthesis of N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-(2-N-(benzyloxycarbonyl)isocytosin-5-ylacetyl)glycine monomer and its incorporation into a PNA molecule via automated Fmoc solid-phase chemistry is described.


Subject(s)
Cytosine/analogs & derivatives , Cytosine/chemistry , Cytosine/metabolism , Fluorenes , Peptide Nucleic Acids/chemical synthesis , Automation , Chemical Phenomena , Chemistry , Chromatography, High Pressure Liquid , Mass Spectrometry , Peptide Nucleic Acids/chemistry , Peptide Nucleic Acids/metabolism
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