Hypoxia inducible factor (HIF) transcription factors reside at the center of signaling pathways used by mammalian cells to sense and respond to low oxygen levels. While essential to maintain oxygen homeostasis, misregulation of HIF protein activity correlates with tumor development and metastasis. To provide artificial routes to target misregulated HIF activity, we identified small molecule antagonists of the HIF-2 transcription factor that bind an internal cavity within the C-terminal PAS domain of the HIF-2α subunit. Here we describe a new class of chiral small molecule ligands that provide the highest affinity binding, the most effective, isoform-selective inhibition of HIF-2 in cells, and trigger the largest protein conformation changes reported to date. The current results further illuminate the molecular mechanism of HIF-2 antagonism and suggest additional routes to develop higher affinity and potency HIF-2 antagonists.

An electrochemical method to synthesize the core macrolactam of diazonamides is described. Large ring-forming dehydrogenation is initiated by anodic oxidation at a graphite surface. The reaction requires no tailoring of the substrate and occurs at ambient temperature in aqueous DMF in an undivided cell open to air. This unique chemistry has enabled a concise, scalable preparation of DZ-2384; a refined analog of diazonamide A slated for clinical development as a cancer therapeutic.

The primary photochemical event in rhodopsin is an l1-cis to 11-trans photoisomerization of its retinylidene chromophore to form the primary intermediate photorhodopsin. Earlier picosecond studies have shown that no intermediate is formed when the retinal li-ene is fixed through a bridging five-membered ring, whereas a photorhodopsin-like intermediate is formed when it is fixed through a flexible seven-membered ring. Results from a rhodopsin analog formed from a retinal with locked l1-ene structure through the more flexible eight-membered ring (Ret8) are described. Incubation of bovine opsin with Ret8 formed two pigments absorbing at 425 nm (P425) and 500 nm (P500). P425, however, is an artifact because it formed from thermally denatured opsin or other proteins and Ret8. Excitation of P500 with a picosecond green pulse led to formation of two intermediates corresponding to photo-and bathorhodopsins. These results demonstrate that an appearance of early intermediates is dependent on the flexibility of the ll-ene and that the photoisomerization of P500 proceeds by stepwise changes of chromophore-protein interaction, which in turn leads to a relaxation of the highly twisted all-trans-retinylidene chromophore in photorhodopsin.The rod photoreceptor cell, responsible for scotopic vision, is a highly sensitive biological photosensor reflecting the high photosensitivity of the visual pigment rhodopsin. Rhodopsin contains an 11-cis-retinal chromophore ( Fig. 1) bound via a protonated Schiff base linkage to Lys-296 of the apoprotein opsin. Since the initial event of rhodopsin after absorption of a photon is a cis-to-trans isomerization of the chromophore (1) with a high quantum yield (2) The conformation of the retinylidene chromophore of bathorhodopsin had been speculated as a twisted all-trans form (4). This was confirmed by application of low temperature CD (8-10), laser Raman (11), and Fourier transform IR (12) spectroscopies. Since photorhodopsin cannot be stabilized at low temperature, its chromophore conformation could not be investigated by means of low temperature spectroscopies applied for that of bathorhodopsin. Therefore, it was investigated by means of picosecond laser photolysis with the aid of rhodopsin analogs (Rh7 and RhS), each of which has a chromophore (Ret7 or Ret5; sbe Fig. 1) that locked the 11-ene in a cis configuration with the trimethylene or methylene bridge, respectively (13)(14)(15). The two retinal analogs differ in flexibility of the ring around the 11-ene from each other. Namely, the seven-membered ring in Ret7 is relatively flexible, whereas the five-membered ring in Ret5 is held in a rigid planar structure. The excitation of Rh7 gave rise to an intermediate corresponding to photorhodopsin, whereas that of Rh5 led only to an excited state (15). Thus, it is suggested that the conversion of rhodopsin to photorhodopsin is due to the twist of the 11-ene and nearby single bonds and resulted in formation of a highly twisted all-trans chromophore. On the other hand, the excitation of Rh...

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.