NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo.

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation in vitro and in vivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration in vitro and in vivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Structure-Based Design and Preclinical Characterization of Selective and Orally Bioavailable Factor XIa Inhibitors: Demonstrating the Power of an Integrated S1 Protease Family Approach.

The serine protease factor XI (FXI) is a prominent drug target as it holds promise to deliver efficacious anticoagulation without an enhanced risk of major bleeds. Several efforts have been described targeting the active form of the enzyme, FXIa. Herein, we disclose our efforts to identify potent, selective, and orally bioavailable inhibitors of FXIa. Compound 1, identified from a diverse library of internal serine protease inhibitors, was originally designed as a complement factor D inhibitor and exhibited submicromolar FXIa activity and an encouraging absorption, distribution, metabolism, and excretion (ADME) profile while being devoid of a peptidomimetic architecture. Optimization of interactions in the S1, S1ß, and S1' pockets of FXIa through a combination of structure-based drug design and traditional medicinal chemistry led to the discovery of compound 23 with subnanomolar potency on FXIa, enhanced selectivity over other coagulation proteases, and a preclinical pharmacokinetics (PK) profile consistent with bid dosing in patients.

Structure-based design and synthesis of macrocyclic human rhinovirus 3C protease inhibitors.

The design and synthesis of macrocyclic inhibitors of human rhinovirus 3C protease is described. A macrocyclic linkage of the P1 and P3 residues, and the subsequent structure-based optimization of the macrocycle conformation and size led to the identification of a potent biochemical inhibitor 10 with sub-micromolar antiviral activity.

DPP9 enzyme activity controls survival of mouse migratory tongue muscle progenitors and its absence leads to neonatal lethality due to suckling defect.

Dipeptidyl peptidase 9 (DPP9) is an intracellular N-terminal post-proline-cleaving enzyme whose physiological function remains largely unknown. We investigated the role of DPP9 enzyme in vivo by characterizing knock-in mice expressing a catalytically inactive mutant form of DPP9 (S729A; DPP9ki/ki mice). We show that DPP9ki/ki mice die within 12-18h after birth. The neonatal lethality can be rescued by manual feeding, indicating that a suckling defect is the primary cause of neonatal lethality. The suckling defect results from microglossia, and is characterized by abnormal formation of intrinsic muscles at the distal tongue. In DPP9ki/ki mice, the number of occipital somite-derived migratory muscle progenitors, forming distal tongue intrinsic muscles, is reduced due to increased apoptosis. In contrast, intrinsic muscles of the proximal tongue and extrinsic tongue muscles, which derive from head mesoderm, develop normally in DPP9ki/ki mice. Thus, lack of DPP9 activity in mice leads to impaired tongue development, suckling defect and subsequent neonatal lethality due to impaired survival of a specific subset of migratory tongue muscle progenitors.

Trypsin inhibitors for the treatment of pancreatitis.

Proline-based trypsin inhibitors occupying the S1-S2-S1' region were identified by an HTS screening campaign. It was discovered that truncation of the P1' moiety and appropriate extension into the S4 region led to highly potent trypsin inhibitors with excellent selectivity against related serine proteases and a favorable hERG profile.

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective, orally available inhibitors of dipeptidyl peptidase IV.

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.

Novel heterocyclic DPP-4 inhibitors for the treatment of type 2 diabetes.

Novel deazaxanthine-based DPP-4 inhibitors have been identified that are potent (IC(50) <10nM) and highly selective versus other dipeptidyl peptidases. Their synthesis and SAR are reported, along with initial efforts to improve the PK profile through decoration of the deazaxanthine core. Optimisation of compound 3a resulted in the identification of compound (S)-4i, which displayed an improved in vitro and ADME profile. Further enhancements to the PK profile were possible by changing from the deazahypoxanthine to the deazaxanthine template, culminating in compound 12g, which displayed good ex vivo DPP-4 inhibition and a superior PK profile in rat, suggestive of once daily dosing in man.

A scalable synthesis of an azabicyclooctanyl derivative, a novel DPP-4 inhibitor.

A practical synthetic strategy to a chiral azabicycclooctanyl derivative (1), a potent DPP-4 inhibitor, starting from a commercially available nortropine is described. The stereogenic center of 1 was established employing a modified protocol of Ellman's diastereoselective addition of a benzylic nucleophile to tert-butanesulfinimine. Other key steps include Corey-Chaykovsky reaction, Meinwald rearrangement, and CDMT-promoted amide bond formation involving a sterically hindered amine 2.

In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics.

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.

Interaction of alpha-and beta-oligoarginine-acids and amides with anionic lipid vesicles: a mechanistic and thermodynamic study.

The interaction of alpha- and beta-oligoarginine amides and acids and of alpha-polyarginine with anionic lipid vesicles was studied. The beta-oligoarginines used were beta3-homologues of the alpha-oligoarginines. Lipid bilayers were composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) containing 5 mol % pyrene-PG (1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-[phospho-rac-1-glycerol]). Kinetic analysis of the binding process onto large unilamellar POPC/POPG (3:7, molar ratio) vesicles (100 nm diameter) shows biphasic time courses for all tested peptides. The first binding step is fast and takes place within approximately 10 s with no disruption of the membrane as indicated by corresponding calcein release measurements. The second binding phase is slow and occurs within the next 30-300 s with substantial membrane disruption. In this context, beta-hexa- and octaarginine amides possess higher second half-times than the beta-hexa- and octaarginine acids of the same chain length. Furthermore beta-octaarginine amide induces a calcein release approximately twice as large as that of the beta-octaarginine acid. Thermodynamic analysis of the binding process, using the complex formation model that assumes that each peptide binds independently to n POPG lipids, reveals apparent binding constants (K(app1)) of approximately 5 x 10(6)-10(8) M(-1) and n-values from 3.7 for beta-hexaarginine acid up to 24.8 for alpha-polyarginine. Although the K(app1)-values are similar, the number of binding sites clearly depends on the chemical nature of the oligoarginine: beta-oligoarginine amides and alpha-oligoarginine acids interact with more lipids than beta-oligoarginine acids of the same length. Calculation of the electrostatic contribution to the total free energy of binding reveals that for all oligoarginines only 25-30% has electrostatic origin. The remaining approximately 70-75% is nonelectrostatic, corresponding to hydrogen bonding and/or hydrophobic interactions. From the obtained data, a mechanism is suggested by which oligoarginines interact with anionic vesicles: (1) initial electrostatic interaction that is fast, nonspecific, and relatively weak; (2) nonelectrostatic interaction that is rate-limiting, stronger, and induces bilayer rigidification as well as release of aqueous contents from the vesicles.

A novel beta-peptidyl aminopeptidase (BapA) from strain 3-2W4 cleaves peptide bonds of synthetic beta-tri- and beta-dipeptides.

A novel bacterial strain that was capable of growing on the beta-tripeptide H-betahVal-betahAla-betahLeu-OH as the sole carbon and nitrogen source was isolated from an enrichment culture. On the basis of physiological characterization, partial 16S rRNA sequencing, and fatty acid analysis, strain 3-2W4 was identified as a member of the family Sphingomonadaceae. Growth on the beta-tripeptide and the beta-dipeptide H-betahAla-betahLeu-OH was observed, and emerging metabolites were characterized. Small amounts of a persisting metabolite, the N-acetylated beta-dipeptide, were identified in both media. According to dissolved organic carbon measurements, 74 to 80% of the available carbon was dissimilated. The beta-peptide-degrading enzyme was purified from the crude cell extract of cells from strain 3-2W4 grown on complex medium. The enzyme was composed of two subunits, and the N-terminal sequences of both were determined. With this information, it was possible to identify the complete nucleotide sequence and to deduce the primary structure of the gene bapA. The gene encoded a beta-peptidyl aminopeptidase (BapA) of 402 amino acids that was synthesized as preprotein with a signal sequence of 29 amino acids. The enzyme was cleaved into two subunits (residues 30 to 278 and 279 to 402). It belonged to the N-terminal nucleophile (Ntn) hydrolase superfamily.

N-linked glycosylated beta-peptides are resistant to degradation by glycoamidase A.

Beta-peptides are resistant to degradation by a variety of proteolytic enzymes that rapidly degrade natural alpha-peptides. This is one of many characteristics that make beta-peptides an attractive class of compounds for drug discovery efforts. To further understand the molecular recognition properties of beta-peptides and the ability of enzymes to degrade them, we have synthesized a series of N-linked glycosylated beta- and alpha-peptides, and tested their stability towards a glycosidase. We found that glyco-beta-peptides that contain N-acetylglucosamine (1) or N,N-diacetylchitobiose (2) are completely stable to degradation by glycoamidase A. In comparison, the glyco-alpha-peptides 3 and 4 containing N-acetylglucosamine or N,N-diacetylchitobiose are degraded. Inhibition experiments using increasing concentrations of a glyco-beta-peptide fail to inhibit degradation of the corresponding glyco-alpha-peptide, even when the glyco-beta-peptide is at a 128-fold higher concentration than the glyco-alpha-peptide. Evidently, the glyco-beta-peptides have a much weaker affinity for the active site of the glycosidase than the corresponding glyco-alpha-peptide. These and the results with proteolytic enzymes suggest that the additional CH(2) group introduced into the alpha-amino acid residues causes beta-peptides not to be recognized by hydrolytic enzymes. The results described herein suggest the potential of beta-peptides that are functionalized with carbohydrates for biological and biomedical investigations, without having to be concerned about the carbohydrate being removed.

Interaction of epothilone analogs with the paclitaxel binding site: relationship between binding affinity, microtubule stabilization, and cytotoxicity.

The interactions of epothilone analogs with the paclitaxel binding site of microtubules were studied. The influence of chemical modifications in the C15 side chain and in C12 on binding affinity and microtubule elongation was characterized. Modifications favorable for binding affinity are (1). a thiomethyl group at C21 of the thiazole side chain, (2). a methyl group at C12 in S configuration, (3). a pyridine side chain with C15 in S configuration, and (4). a cyclopropyl moiety between C12 and C13. The same modification in different ligands has similar effect on affinity, allowing good structure-affinity characterization. The correlation between binding, microtubule stabilization, and cytotoxicity of the compounds has been determined, showing differential effects of the modifications. The binding constants correlate well with IC(50) values, demonstrating that affinity measurements are a useful tool for drug design.

Chemical and biological investigations of beta-oligoarginines.

In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-beta3 hArg(Boc)2-OH is used for manual solid-phase synthesis of beta3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, beta3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2-4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-beta2 hArg-OH and (S)-H-beta3 hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the beta3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free beta-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 microM; Table 1), and no significant antibiotic activity (MIC > or = 64 microg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant beta3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain beta-oligoarginine amides (8 and 10 residues; Figs. 4-6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37 degrees and 4 degrees with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the beta-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-microM concentration of the fluorescence-labelled beta-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the beta-octaarginine derivative show migration of the beta-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled beta-octa- and beta-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the beta-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a beta-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the 'protein transduction' by beta-oligoarginines are discussed.