Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

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Lipophilicity has a dominant effect on many parameters that determine unbound drug exposure as well as drug potency. Despite this, analysis of a large body of drug data indicates lipophilicity has no consistent directional impact on dose. This can be rationalized based on the interplay of the effects of lipophilicity on individual parameter values in pharmacokinetic equations. We believe this undermines the effectiveness of strategies that target specific ranges for drug parameters for which lipophilicity plays such a dominant role. As a result, our research organization no longer leverages the common approach of screening for low intrinsic clearance in vitro to target high unbound exposure in vivo. Instead, we advocate for approaches less biased to lipophilicity through optimization of key parameter ratios controlling dose. We believe this improves efficiency in drug discovery by enabling exploration of broad physicochemical space.

The annexins are water soluble proteins possessing a hydrophilic surface, which belong to a family of proteins which (a) bind ('annex') both calcium and phospholipids, and (b) form voltage-dependent calcium channels within planar lipid bilayers. Annexins types are diverse (94 annexins in 45 species) and they belong to an enormous multigene family that ranges throughout all eukaryotic kingdoms. Although the structure of these proteins is now well known their functional and physiological roles remain largely unknown and circumstantial. Various experimental approaches provided evidence that annexins function as Ca(2+) channels that could act as regulators of membrane fusion. The identity of annexins is derived from the conserved 34 kDa C-terminal domain which comprises four repeats - except for annexin VI, with eight repeats - of a sequence of approximately seventy amino acids, which holds the area known as the 'endonexin fold', with its identifying GXGTDE. Annexins have been placed into three subgroups of (1) tetrad core and short amino terminal, (2) tetrad core and long amino terminal, and (3) octad core and short amino terminal. The repeats are highly conserved, each forming a compact alpha-helical domain comprising five alpha-helices wound in a right-handed superhelix. Four domains are formed, arranged in a nearly flat and cyclical array, with domains I and IV, and II and III respectively forming two tightly organised modules with almost twofold symmetry. A hydrophilic pore lies at the centre of the molecule, forming a prominent ion channel coated with charged and highly conserved residues. The annexin molecule is slightly curved, with both a convex and a concave face. The cation/anion permeability ratios and the selectivity sequence of the ion channels formed by several annexins confirm the selectivity of the annexins for Ca(2+) over other divalent cations, and reveals the importance of structural sites, e.g. amino acid positions 17, 78, 95 and 112 for the identification of the ion channel's position, function and regulation. Some are sensitive to low doses of the phenothiazine drugs, trifluoperazine (an anti-schizophrenia drug) and promethazine (anti nausea drug) La(3+) and Cd(2+), (blockers of voltage-gated Ca(2+) channels) nifedipine (an inhibitor of non-activating Ca(2+) channels). There are two main competing models used to explain in vitro ion channel activity of annexins: one involves changes in the conductance of ion via electrostatic disturbance of the membrane surface; the other involves a much more extensive alteration in protein structure and a correspondingly deeper penetration into the membrane.

Despite their proven antidiabetic efficacy, widespread use of peroxisome proliferator-activated receptor (PPAR)␥ agonists has been limited by adverse cardiovascular effects. To overcome this shortcoming, selective PPAR␥ modulators (SPPAR␥Ms) have been identified that have antidiabetic efficacy comparable with full agonists with improved tolerability in preclinical species. The results of structural studies support the proposition that SPPAR␥Ms interact with PPAR␥ differently from full agonists, thereby providing a physical basis for their novel activities. Herein, we describe a novel PPAR␥ ligand, SPPAR␥M2. This compound was a partial agonist in a cell-based transcriptional activity assay, with diminished adipogenic activity and an attenuated gene signature in cultured human adipocytes. X-ray cocrystallography studies demonstrated that, unlike rosiglitazone, SPPAR␥M2 did not interact with the Tyr473 residue located within helix 12 of the ligand binding domain (LBD). Instead, SPPAR␥M2 was found to bind to and activate human PPAR␥ in which the Tyr473 residue had been mutated to alanine (hPPAR␥Y473A), with potencies similar to those observed with the wild-type receptor (hPPAR␥WT). In additional studies, we found that the intrinsic binding and functional potencies of structurally distinct SPPAR␥Ms were not diminished by the Y473A mutation, whereas those of various thiazolidinedione (TZD) and non-TZD PPAR␥ full agonists were reduced in a correlative manner. These results directly demonstrate the important role of Tyr473 in mediating the interaction of full agonists but not SPPAR␥Ms with the PPAR␥ LBD, thereby providing a precise molecular determinant for their differing pharmacologies.The peroxisome proliferator-activated receptors (PPARs) ␥, ␦, and ␣ compose a nuclear receptor subfamily that modulates the transcription of a large compendium of genes encoding proteins that regulate lipid metabolism, cell differentiation, and signal transduction in a ligand-dependent manner (Berger and Moller, 2002). PPAR␥ has been shown to be a master regulator of adipogenesis and nutrient metabolism in adipocytes where it is highly expressed. Thiazolidinedione (TZD) PPAR␥ full agonists have demonstrated clinical efficacy for the treatment of type 2 diabetes mellitus (T2DM) patients . However, the use of these insulin-sensitizing agents has been restricted because of their association with several adverse effects, including

Previous studies demonstrated that the naturally occurring electrophile and PPARgamma ligand, nitrolinoleic acid (NO(2)-LA), exists as a mixture of four regioisomers [Alexander, R. L., et al. (2006) Biochemistry 45, 7889-7896]. We hypothesized that these alternative isomers have distinct bioactivities; therefore, to determine if the regioisomers are quantitatively or qualitatively different with respect to PPARgamma activation, NO(2)-LA was separated into three fractions which were identified by NMR (13-NO(2)-LA, 12-NO(2)-LA, and a mixture of 9- and 10-NO(2)-LA) and characterized for PPARgamma interactions. A competition radioligand binding assay showed that all three NO(2)-LA fractions had similar binding affinities for PPARgamma (IC(50) = 0.41-0.60 microM) that were comparable to that of the pharmaceutical ligand, rosiglitazone (IC(50) = 0.25 microM). However, when PPARgamma-dependent transcription activation was examined, there were significant differences observed among the NO(2)-LA fractions. Each isomer behaved as a partial agonist in this reporter gene assay; however, the 12-NO(2) derivative was the most potent with respect to maximum activation of PPARgamma and an EC(50) of 0.045 microM (compare with the rosiglitazone EC(50) of 0.067 microM), while the 13-NO(2) and 9- and 10-NO(2) derivatives were considerably less effective with EC(50) values of 0.41-0.62 microM. We conclude that the regioisomers of NO(2)-LA are not functionally equivalent. The 12-NO(2) derivative appears to be the most potent in PPARgamma-dependent transcription activation, whereas the weaker PPARgamma agonists, 13-NO(2) and 9- and 10-NO(2), may be relatively more important in signaling via other, PPARgamma-independent pathways in which this family of nitrolipid electrophiles is implicated.

The leucine-rich repeat kinase 2 (LRRK2) protein has been genetically and functionally linked to Parkinson’s disease (PD), a disabling and progressive neurodegenerative disorder whose current therapies are limited in scope and efficacy. In this report, we describe a rigorous hit-to-lead optimization campaign supported by structural enablement, which culminated in the discovery of brain-penetrant, candidate-quality molecules as represented by compounds 22 and 24. These compounds exhibit remarkable selectivity against the kinome and offer good oral bioavailability and low projected human doses. Furthermore, they showcase the implementation of stereochemical design elements that serve to enable a potency- and selectivity-enhancing increase in polarity and hydrogen bond donor (HBD) count while maintaining a central nervous system-friendly profile typified by low levels of transporter-mediated efflux and encouraging brain penetration in preclinical models.

Nitroalkene fatty acids are potent endogenous ligand activators of PPARγ-dependent transcription. Previous studies with the naturally occurring regioisomers of nitrolinoleic acid revealed that the isomers are not equivalent with respect to PPARγ activation. To gain further insight into the structureactivity relationships between nitroalkenes and PPARγ, we examined additional naturally occurring nitroalkenes derived from oleic acid, 9-nitrooleic acid (E-9-NO 2 -18:1 [1]) and 10-nitrooleic acid (E-10-NO 2 -18:1 [2]), and several synthetic nitrated enoic fatty acids of variable carbon chain length, double bonds, and nitration site. At submicromolar concentrations, E-12-NO 2 derivatives were considerably more potent than isomers nitrated at carbons 5, 6, 9, 10 or 13; and, chain length (16 versus 18) or number of double bonds (one versus two) was of little consequence for PPARγ activation. Interestingly, at higher concentrations (> 2 μM) the nitrated enoic fatty acids (E-9-NO 2 -18:1 [1], E-9-NO 2 -16:1 [3], E-10-NO 2 -18:1 [2], and E-12-NO 2 -18:1 [7]) deviated significantly from the saturable pattern of PPARγ activation observed for nitrated 1,4-dienoic fatty acids