A general method to design dominant negatives to B-HLHZip proteins that abolish DNA binding

We describe a method to design dominant-negative proteins (D-N) to the basic helix–loop–helix–leucine zipper (B-HLHZip) family of sequence-specific DNA binding transcription factors. The D-Ns specifically heterodimerize with the B-HLHZip dimerization domain of the transcription factors and abolish DNA binding in an equimolar competition. Thermal denaturation studies indicate that a heterodimer between a Myc B-HLHZip domain and a D-N consisting of a 12-amino acid sequence appended onto the Max dimerization domain (A-Max) is −6.3 kcal⋅mol−1 more stable than the Myc:Max heterodimer. One molar equivalent of A-Max can totally abolish the DNA binding activity of a Myc:Max heterodimer. This acidic extension also has been appended onto the dimerization domain of the B-HLHZip protein Mitf, a member of the transcription factor enhancer binding subfamily, to produce A-Mitf. The heterodimer between A-Mitf and the B-HLHZip domain of Mitf is −3.7 kcal⋅mol−1 more stable than the Mitf homodimer. Cell culture studies show that A-Mitf can inhibit Mitf-dependent transactivation both in acidic extension and in a dimerization-dependent manner. A-Max can inhibit Myc-dependent foci formation twice as well as the Max dimerization domain (HLHZip). This strategy of producing D-Ns may be applicable to other B-HLHZip or B-HLH proteins because it provides a method to inhibit the DNA binding of these transcription factors in a dimerization-specific manner.

A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.

Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se--i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its gene-delivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

Design and biological activities of L-163,191 (MK-0677): a potent, orally active growth hormone secretagogue.

A potent, orally active growth hormone (GH) secretagogue L-163,191 belonging to a recently synthesized structural class has been characterized. L-163,191 releases GH from rat pituitary cells in culture with EC50 = 1.3 +/- 0.09 nM and is mechanistically indistinguishable from the GH-releasing peptide GHRP-6 and the prototypical nonpeptide GH secretagogue L-692,429 but clearly distinguishable from the natural GH secretagogue, GH-releasing hormone. L-163,191 elevates GH in dogs after oral doses as low as 0.125 mg/kg and was shown to be specific in its release of GH without significant effect on plasma levels of aldosterone, luteinizing hormone, thyroxine, and prolactin after oral administration of 1 mg/kg. Only modest increases in cortisol were observed. Based on these properties, L-163,191 has been selected for clinical studies.