Pharmacological inhibition of the cysteine protease cathepsin C improves graft function after heart transplantation in rats.

BACKGROUND: Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX. METHODS: In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis. RESULTS: The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX. CONCLUSIONS: We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.

Molecular basis of TASL recruitment by the peptide/histidine transporter 1, PHT1.

PHT1 is a histidine /oligopeptide transporter with an essential role in Toll-like receptor innate immune responses. It can act as a receptor by recruiting the adaptor protein TASL which leads to type I interferon production via IRF5. Persistent stimulation of this signalling pathway is known to be involved in the pathogenesis of systemic lupus erythematosus (SLE). Understanding how PHT1 recruits TASL at the molecular level, is therefore clinically important for the development of therapeutics against SLE and other autoimmune diseases. Here we present the Cryo-EM structure of PHT1 stabilized in the outward-open conformation. By combining biochemical and structural modeling techniques we propose a model of the PHT1-TASL complex, in which the first 16 N-terminal TASL residues fold into a helical structure that bind in the central cavity of the inward-open conformation of PHT1. This work provides critical insights into the molecular basis of PHT1/TASL mediated type I interferon production.

BI 1291583: a novel selective inhibitor of cathepsin C with superior in vivo profile for the treatment of bronchiectasis.

BACKGROUND: Airway inflammation in chronic inflammatory lung diseases (e.g. bronchiectasis) is partly mediated by neutrophil-derived serine protease (NSP)/antiprotease imbalance. NSPs are activated during neutrophil myelopoiesis in bone marrow by cathepsin C (CatC; DPP1). CatC is therefore an attractive target to reduce NSP activity in the lungs of patients with bronchiectasis, restoring the protease/antiprotease balance. We report results from the preclinical pharmacological assessment of the novel CatC inhibitor BI 1291583. METHODS: Binding kinetics of BI 1291583 to human CatC were determined by surface plasmon resonance. In vitro inhibition of human CatC activity was determined by CatC-specific fluorescent assay, and selectivity was assessed against related cathepsins and unrelated proteases. Inhibition of NSP neutrophil elastase (NE) production was assessed in a human neutrophil progenitor cell line. In vivo inhibition of NE and NSP proteinase 3 (PR3) in bronchoalveolar lavage fluid (BALF) neutrophils after lipopolysaccharide (LPS) challenge and distribution of BI 1291583 was determined in a mouse model. RESULTS: BI 1291583 bound human CatC in a covalent, reversible manner, selectively and fully inhibiting CatC enzymatic activity. This inhibition translated to concentration-dependent inhibition of NE activation in U937 cells and dose-dependent, almost-complete inhibition of NE and PR3 activity in BALF neutrophils in an in vivo LPS-challenge model in mice. BI 1291583 exhibited up to 100 times the exposure in the target tissue bone marrow compared with plasma. CONCLUSION: BI 1291583-mediated inhibition of CatC is expected to restore the protease-antiprotease balance in the lungs of patients with chronic airway inflammatory diseases such as bronchiectasis.

BIreactive: Expanding the Scope of Reactivity Predictions to Propynamides.

We present the first comprehensive study on the prediction of reactivity for propynamides. Covalent inhibitors like propynamides often show improved potency, selectivity, and unique pharmacologic properties compared to their non-covalent counterparts. In order to achieve this, it is essential to tune the reactivity of the warhead. This study shows how three different in silico methods can predict the in vitro properties of propynamides, a covalent warhead class integrated into approved drugs on the market. Whereas the electrophilicity index is only applicable to individual subclasses of substitutions, adduct formation and transition state energies have a good predictability for the in vitro reactivity with glutathione (GSH). In summary, the reported methods are well suited to estimate the reactivity of propynamides. With this knowledge, the fine tuning of the reactivity is possible which leads to a speed up of the design process of covalent drugs.

Targeting Cathepsin C in PR3-ANCA Vasculitis.

BACKGROUND: The ANCA autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO) are exclusively expressed by neutrophils and monocytes. ANCA-mediated activation of these cells is the key driver of the vascular injury process in ANCA-associated vasculitis (AAV), and neutrophil serine proteases (NSPs) are disease mediators. Cathepsin C (CatC) from zymogens activates the proteolytic function of NSPs, including PR3. Lack of NSP zymogen activation results in neutrophils with strongly reduced NSP proteins. METHODS: To explore AAV-relevant consequences of blocking NSP zymogen activation by CatC, we used myeloid cells from patients with Papillon-Lefèvre syndrome, a genetic deficiency of CatC, to assess NSPs and NSP-mediated endothelial cell injury. We also examined pharmacologic CatC inhibition in neutrophil-differentiated human hematopoietic stem cells, primary human umbilical vein cells, and primary glomerular microvascular endothelial cells. RESULTS: Patients with Papillon-Lefèvre syndrome showed strongly reduced NSPs in neutrophils and monocytes. Neutrophils from these patients produced a negative PR3-ANCA test, presented less PR3 on the surface of viable and apoptotic cells, and caused significantly less damage in human umbilical vein cells. These findings were recapitulated in human stem cells, in which a highly specific CatC inhibitor, but not prednisolone, reduced NSPs without affecting neutrophil differentiation, reduced membrane PR3, and diminished neutrophil activation upon PR3-ANCA but not MPO-ANCA stimulation. Compared with healthy controls, neutrophils from patients with Papillon-Lefèvre syndrome transferred less proteolytically active NSPs to glomerular microvascular endothelial cells, the cell type targeted in ANCA-induced necrotizing crescentic glomerulonephritis. Finally, both genetic CatC deficiency and pharmacologic inhibition, but not prednisolone, reduced neutrophil-induced glomerular microvascular endothelial cell damage. CONCLUSIONS: These findings may offer encouragement for clinical studies of adjunctive CatC inhibitor in patients with PR3-AAV.

Covalent inhibitor reactivity prediction by the electrophilicity index-in and out of scope.

Drug discovery is an expensive and time-consuming process. To make this process more efficient quantum chemistry methods can be employed. The electrophilicity index is one property that can be calculated by quantum chemistry methods, and if calculated correctly gives insight into the reactivity of covalent inhibitors. Herein we present the usage of the electrophilicity index on three common warheads, i.e., acrylamides, 2-chloroacetamides, and propargylamides. We thoroughly examine the properties of the electrophilicity index, show which pitfalls should be avoided, and what the requirements to successfully apply the electrophilicity index are.

BIreactive: A Machine-Learning Model to Estimate Covalent Warhead Reactivity.

In the past decade, the pharmaceutical industry has paid closer attention to covalent drugs. Differently from standard noncovalent drugs, these compounds can exhibit peculiar properties, such as higher potency or longer duration of target inhibition with a potentially lower dosage. These properties are mainly driven by the reactive functional group present in the compound, the so-called warhead that forms a covalent bond with a specific nucleophilic amino-acid on the target. In this work, we report the possibility to combine ab initio activation energies with machine-learning to estimate covalent compound intrinsic reactivity. The idea behind this approach is to have a precise estimation of the transition state barriers, and thus of the compound reactivity, but with the speed of a machine-learning algorithm. We call this method "BIreactive". Here, we demonstrate this approach on acrylamides and 2-chloroacetamides, two warhead classes that possess different reaction mechanisms. In combination with our recently implemented truncation algorithm, we also demonstrate the possibility to use BIreactive not only for fragments but also for lead-like molecules. The generic nature of this approach allows also the extension to several other warheads. The combination of these factors makes BIreactive a valuable tool for the covalent drug discovery process in a pharmaceutical context.

A Fast Ab Initio Predictor Tool for Covalent Reactivity Estimation of Acrylamides.

Thanks to their unique mode of action, covalent drugs represent an exceptional opportunity for drug design. After binding to a biologically relevant target system, covalent compounds form a reversible or irreversible covalent bond with a nucleophilic amino acid. Due to the inherently large binding energy of a covalent bond, covalent binders exhibit higher potencies and thus allow potentially lower drug dosages. However, a proper balancing of compound reactivity is key for the design of covalent binders, to achieve high levels of target inhibition while minimizing promiscuous covalent binding to nontarget proteins. In this work, we demonstrated the possibility to apply the electrophilicity index concept to estimate covalent compound reactivity. We tested this approach on acrylamides, one of the most prominent classes of covalent warheads. Our study clearly demonstrated that, for compounds with molecular weight (MW) below 250 Da, the electrophilicity index can be directly used to estimate compound reactivity. On the other hand, for leadlike molecules (MW > 250 Da) we implemented a new truncation algorithm that has to be applied before reactivity calculations. This algorithm can ensure the localization of HOMO/LUMO orbitals on the compound warhead and thus a correct estimation of its reactivity. Our results also indicate that caution should be used when employing the electrophilicity index to estimate the reactivity of nonterminal acrylamides. The nonparametric nature of this method and its reasonable computational cost make it a suitable tool to support covalent drug design.

Allosteric Activation of Striatal-Enriched Protein Tyrosine Phosphatase (STEP, PTPN5) by a Fragment-like Molecule.

Protein tyrosine phosphatase non-receptor type 5 (PTPN5, STEP) is a brain specific phosphatase that regulates synaptic function and plasticity by modulation of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking. Dysregulation of STEP has been linked to neurodegenerative and neuropsychiatric diseases, highlighting this enzyme as an attractive therapeutic target for drug discovery. Selective targeting of STEP with small molecules has been hampered by high conservation of the active site among protein tyrosine phosphatases. We report the discovery of the first small molecule allosteric activator for STEP that binds to the phosphatase domain. Allosteric binding is confirmed by both X-ray and 15N NMR experiments, and specificity has been demonstrated by an enzymatic test cascade. Molecular dynamics simulations indicate stimulation of enzymatic activity by a long-range allosteric mechanism. To allow the scientific community to make use of this tool, we offer to provide the compound in the course of an open innovation initiative.

Rodent selectivity of piperidine-4-yl-1H-indoles, a series of CC chemokine receptor-3 (CCR3) antagonists: insights from a receptor model.

Rodent selectivity data of piperidine-4-yl-1H-indoles, a series of CC chemokine receptor-3 (CCR3) antagonists, are presented and discussed as part of an overall optimization effort within this lead compound class. Although attachment of an acidic moiety to the 1-position of the indole led to an overall balanced in vitro profile, in particular reducing inhibition of the hERG channel, potency on the rat and mouse receptor worsened. These findings could be rationalized in the context of a CCR3 homology model.

Bioconjugates of enantiomerically pure organopalladium compounds by metal-assisted positional selective transesterifications at palladium enolates.

The synthesis of enantiomerically pure palladatricyclo[4.1.0.0(2,4)]heptanes and their modification by an unprecedented and very efficient positional selective transesterification is described. The mild reaction conditions are most probably based on an acceleration of the transesterification due to assistance by the metal. This novel approach allows straightforward access to a large number of structurally diverse organometallic complexes. The functional groups on the newly installed ester moieties were modified by using standard peptide synthesis protocols, Sonogashira reactions, and nucleophilic substitution reactions. The cellular uptake of these organometallic species was traced by confocal microscopy and their biological activity was evaluated by using different cell lines. Inhibition of cell growth and induction of apoptotic cell death by these novel palladium heterocycles are equivalent to Cisplatin.

Functional interaction of metabotropic glutamate receptor 5 and NMDA-receptor by a metabotropic glutamate receptor 5 positive allosteric modulator.

The NMDA (N-methyl-D-aspartate)-receptor is fundamentally involved in cognitive functions. Recent studies demonstrated a functional interaction between the metabotropic glutamate receptor 5 (mGlu(5) receptor) and the NMDA-receptor in neurons. In rat hippocampal slices, it was shown that activation of mGlu(5) receptor by a positive modulator in the presence of a subthreshold agonist concentration potentiated NMDA-receptor mediated currents and phosphorylation of intracellular signalling proteins. In the present study, we investigated the functional interaction of mGlu(5) receptor and NMDA-receptor by the selective mGlu(5) receptor positive modulator ADX-47273 in-vitro and in-vivo. In rat primary neurons, this compound potentiated Ca(2+) mobilization in the presence of a subthreshold concentration of the mGluR(1/5) agonist DHPG (0.3 microM) with an EC(50) of 0.28+/-0.05 microM. NMDA-induced Ca(2+)-mobilization in primary neurons could be potentiated when neurons were pre-stimulated with 1 microM ADX-47273 in the presence of 0.3 microM DHPG. The specific mGlu(5) receptor antagonist MPEP and the Src-family kinase inhibitor PP2 blocked this potentiation demonstrating the functional interaction of the NMDA-receptor and mGlu(5) receptor in neurons. Furthermore, ADX-47273 elicited an enhancement of NMDA-receptor dependent long-term potentiation in rat hippocampal slices that could be reversed by MPEP. After intraperitoneal administration to rats, ADX-47273 showed a dose-dependent reduction of NMDA-receptor antagonist (ketamine) induced hyperlocomotion, supporting the mechanistic interaction of the NMDA-receptor and mGlu(5) receptor in-vivo. In conclusion, these findings further support the idea of a functional interaction between the mGlu(5) receptor and NMDA-receptor, which may provide a pharmacological strategy for addressing CNS diseases with cognitive impairments linked to NMDA-receptor hypofunction.

Development of a Friedel-Crafts triflation.

The development of a new variant of the Friedel-Crafts reaction that yields 3-aryl enol triflates is described. The reaction is practical, is atom-economical, and works well with electron-rich arene substrates. [reaction: see text].

Synthetic studies toward the haouamines.

[reaction: see text] A concise synthetic approach toward the haouamines based on Stork-Danheiser alkylation and Friedel-Crafts chemistry is described. A novel electrophilic aromatic substitution with concomitant formation of an enol triflate is reported.

An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs.

Riboswitches are highly structured RNA elements that control the expression of many bacterial genes by binding directly to small metabolite molecules with high specificity and affinity. In Bacillus subtilis, two classes of riboswitches have been described that discriminate between guanine and adenine despite an extremely high degree of homology both in their primary and secondary structure. We have identified intermolecular base triples between both purine ligands and their respective riboswitch RNAs by NMR spectroscopy. Here, specificity is mediated by the formation of a Watson-Crick base pair between the guanine ligand and a C residue or the adenine ligand and a U residue of the cognate riboswitch RNA, respectively. In addition, a second base-pairing interaction common to both riboswitch purine complexes involves a uridine residue of the RNA and the N3/N9 edge of the purine ligands. This base pairing is mediated by a previously undescribed hydrogen-bonding scheme that contributes to the affinity of the RNA-ligand interaction. The observed intermolecular hydrogen bonds between the purine ligands and the RNA rationalize the previously observed change in specificity upon a C to U mutation in the core of the purine riboswitch RNAs and the differences in the binding affinities for a number of purine analogs.