Conditional Cell-Penetrating Peptide Exposure as Selective Nanoparticle Uptake Signal.

A major bottleneck diminishing the therapeutic efficacy of various drugs is that only small proportions of the administered dose reach the site of action. One promising approach to increase the drug amount in the target tissue is the delivery via nanoparticles (NPs) modified with ligands of cell surface receptors for the selective identification of target cells. However, since receptor binding can unintentionally trigger intracellular signaling cascades, our objective was to develop a receptor-independent way of NP uptake. Cell-penetrating peptides (CPPs) are an attractive tool since they allow efficient cell membrane crossing. So far, their applicability is severely limited as their uptake-promoting ability is nonspecific. Therefore, we aimed to achieve a conditional CPP-mediated NP internalization exclusively into target cells. We synthesized different CPP candidates and investigated their influence on nanoparticle stability, ζ-potential, and uptake characteristics in a core-shell nanoparticle system consisting of poly(lactid-co-glycolid) (PLGA) and poly(lactic acid)-poly(ethylene glycol) (PLA10kPEG2k) block copolymers with CPPs attached to the PEG part. We identified TAT47-57 (TAT) as the most promising candidate and subsequently combined the TAT-modified PLA10kPEG2k polymer with longer PLA10kPEG5k polymer chains, modified with the potent angiotensin-converting enzyme 2 (ACE2) inhibitor MLN-4760. While MLN-4760 enables selective target cell identification, the additional PEG length hides the CPP during a first unspecific cell contact. Only after the previous selective binding of MLN-4760 to ACE2, the established spatial proximity exposes the CPP, triggering cell uptake. We found an 18-fold uptake improvement in ACE2-positive cells compared to unmodified particles. In summary, our work paves the way for a conditional and thus highly selective receptor-independent nanoparticle uptake, which is beneficial in terms of avoiding side effects.

Development of Fluorescent AF64394 Analogues Enables Real-Time Binding Studies for the Orphan Class A GPCR GPR3.

The orphan G protein-coupled receptor (oGPCR) GPR3 represents a potential drug target for the treatment of Alzheimer's disease and metabolic disorders. However, the limited toolbox of pharmacological assays hampers the development of advanced ligands. Here, we developed a signaling pathway-independent readout of compound-GPR3 interaction. Starting from computational binding pose predictions of the most potent GPR3 ligand, we designed a series of fluorescent AF64394 analogues and assessed their suitability for BRET-based binding studies. The most potent ligand, 45 (UR-MB-355), bound to GPR3 and closely related receptors, GPR6 and GPR12, with similar submicromolar affinities. Furthermore, we found that 45 engages GPR3 in a distinct mode compared to AF64394, and coincubation studies with the GPR3 agonist diphenyleneiodonium chloride revealed allosteric modulation of 45 binding. These insights provide new cues for the pharmacological manipulation of GPR3 activity. This novel binding assay will foster the development of future drugs acting through these pharmacologically attractive oGPCRs.

Characterization of caspase-2 inhibitors based on specific sites of caspase-2-mediated proteolysis.

Since the discovery of the caspase-2 (Casp2)-mediated ∆tau314 cleavage product and its associated impact on tauopathies such as Alzheimer's disease, the design of selective Casp2 inhibitors has become a focus in medicinal chemistry research. In the search for new lead structures with respect to Casp2 selectivity and drug-likeness, we have taken an approach by looking more closely at the specific sites of Casp2-mediated proteolysis. Using seven selected protein cleavage sequences, we synthesized a peptide series of 53 novel molecules and studied them using in vitro pharmacology, molecular modeling, and crystallography. Regarding Casp2 selectivity, AcITV(Dab)D-CHO (23) and AcITV(Dap)D-CHO (26) demonstrated the best selectivity (1-6-fold), although these trends were only moderate. However, some analogous tetrapeptides, most notably AcDKVD-CHO (45), showed significantly increased Casp3 selectivities (>100-fold). Tetra- and tripeptides display decreased or no Casp2 affinity, supporting the assumption that a motif of five amino acids is required for efficient Casp2 inhibition. Overall, the results provide a reasonable basis for the development of both selective Casp2 and Casp3 inhibitors.

Caspase-2 Inhibitor Blocks Tau Truncation and Restores Excitatory Neurotransmission in Neurons Modeling FTDP-17 Tauopathy.

Synaptic and cognitive deficits mediated by a severe reduction in excitatory neurotransmission caused by a disproportionate accumulation of the neuronal protein tau in dendritic spines is a fundamental mechanism that has been found repeatedly in models of tauopathies, including Alzheimer's disease, Lewy body dementia, frontotemporal dementia, and traumatic brain injury. Synapses thus damaged may contribute to dementia, among the most feared cause of debilitation in the elderly, and currently there are no treatments to repair them. Caspase-2 (Casp2) is an essential component of this pathological cascade. Although it is believed that Casp2 exerts its effects by hydrolyzing tau at aspartate-314, forming Δtau314, it is also possible that a noncatalytic mechanism is involved because catalytically dead Casp2 is biologically active in at least one relevant cellular pathway, that is, autophagy. To decipher whether the pathological effects of Casp2 on synaptic function are due to its catalytic or noncatalytic properties, we discovered and characterized a new Casp2 inhibitor, compound 1 [pKi (Casp2) = 8.12], which is 123-fold selective versus Casp3 and >2000-fold selective versus Casp1, Casp6, Casp7, and Casp9. In an in vitro assay based on Casp2-mediated cleavage of tau, compound 1 blocked the production of Δtau314. Importantly, compound 1 prevented tau from accumulating excessively in dendritic spines and rescued excitatory neurotransmission in cultured primary rat hippocampal neurons expressing the P301S tau variant linked to FTDP-17, a familial tauopathy. These results support the further development of small-molecule Casp2 inhibitors to treat synaptic deficits in tauopathies.

Structure-Based Design and Biological Evaluation of Novel Caspase-2 Inhibitors Based on the Peptide AcVDVAD-CHO and the Caspase-2-Mediated Tau Cleavage Sequence YKPVD314.

Alzheimer's disease (AD) was first described by Alois Alzheimer over 100 years ago, but there is still no overarching theory that can explain its cause in detail. There are also no effective therapies to treat either the cause or the associated symptoms of this devastating disease. A potential approach to better understand the pathogenesis of AD could be the development of selective caspase-2 (Casp2) probes, as we have shown that a Casp2-mediated cleavage product of tau (Δtau314) reversibly impairs cognitive and synaptic function in animal models of tauopathies. In this article, we map out the Casp2 binding site through the preparation and assay of a series of 35 pentapeptide inhibitors with the goal of gaining selectivity against caspase-3 (Casp3). We also employed computational docking methods to understand the key interactions in the binding pocket of Casp2 and the differences predicted for binding at Casp3. Moreover, we crystallographically characterized the binding of selected pentapeptides with Casp3. Furthermore, we engineered and expressed a series of recombinant tau mutants and investigated them in an in vitro cleavage assay. These studies resulted in simple peptidic inhibitors with nanomolar affinity, for example, AcVDV(Dab)D-CHO (24) with up to 27.7-fold selectivity against Casp3. Our findings provide a good basis for the future development of selective Casp2 probes and inhibitors that can serve as pharmacological tools in planned in vivo studies and as lead compounds for the design of bioavailable and more drug-like small molecules.

Cardiac Effects of Novel Histamine H2 Receptor Agonists.

In an integrative approach, we studied cardiac effects of recently published novel H2 receptor agonists in the heart of mice that overexpress the human H2 receptor (H2-TG mice) and littermate wild type (WT) control mice and in isolated electrically driven muscle preparations from patients undergoing cardiac surgery. Under our experimental conditions, the H2 receptor agonists UR-Po563, UR-MB-158, and UR-MB-159 increased force of contraction in left atrium from H2-TG mice with pEC50 values of 8.27, 9.38, and 8.28, respectively, but not in WT mice. Likewise, UR-Po563, UR-MB-158, and UR-MB-159 increased the beating rate in right atrium from H2-TG mice with pEC50 values of 9.01, 9.24, and 7.91, respectively, but not from WT mice. These effects could be antagonized by famotidine, a H2 receptor antagonist. UR-Po563 (1 µM) increased force of contraction in Langendorff-perfused hearts from H2-TG but not WT mice. Similarly, UR-Po563, UR-MB-158, or UR-MB-159 increased the left ventricular ejection fraction in echocardiography of H2-TG mice. Finally, UR-Po563 increased force of contraction in isolated human right atrial muscle strips. The contractile effects of UR-Po563 in H2-TG mice were accompanied by an increase in the phosphorylation state of phospholamban. In summary, we report here three recently developed agonists functionally stimulating human cardiac H2 receptors in vitro and in vivo. We speculate that these compounds might be of some merit to treat neurologic disorders if their cardiac effects are blocked by concomitantly applied receptor antagonists that cannot pass through the blood-brain barrier or might be useful to treat congestive heart failure in patients. SIGNIFICANCE STATEMENT: Recently, a new generation of histamine H2 receptor (H2R) agonists has been developed as possible treatment option for Alzheimer's disease. Here, possible cardiac (side) effects of these novel H2R agonists have been evaluated.

Abolishing Dopamine D2long/D3 Receptor Affinity of Subtype-Selective Carbamoylguanidine-Type Histamine H2 Receptor Agonists.

3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H2 receptor (H2R) agonists, but their applicability as pharmacological tools to elucidate the largely unknown H2R functions in the central nervous system (CNS) is compromised by their concomitant high affinity toward dopamine D2-like receptors (especially to the D3R). To improve the selectivity, a series of novel carbamoylguanidine-type ligands containing various heterocycles, spacers, and side residues were rationally designed, synthesized, and tested in binding and/or functional assays at H1-4 and D2long/3 receptors. This study revealed a couple of selective candidates (among others 31 and 47), and the most promising ones were screened at several off-target receptors, showing good selectivities. Docking studies suggest that the amino acid residues (3.28, 3.32, E2.49, E2.51, 5.42, and 7.35) are responsible for the different affinities at the H2- and D2long/3-receptors. These results provide a solid base for the exploration of the H2R functions in the brain in further studies.

Pharmacological characterization of a new series of carbamoylguanidines reveals potent agonism at the H2R and D3R.

Even today, the role of the histamine H2 receptor (H2R) in the central nervous system (CNS) is widely unknown. In previous research, many dimeric, high-affinity and subtype-selective carbamoylguanidine-type ligands such as UR-NK22 (5, pKi = 8.07) were reported as H2R agonists. However, their applicability to the study of the H2R in the CNS is compromised by their molecular and pharmacokinetic properties, such as high molecular weight and, consequently, a limited bioavailability. To address the need for more drug-like H2R agonists with high affinity, we synthesized a series of monomeric (thio)carbamoylguanidine-type ligands containing various spacers and side-chain moieties. This structural simplification resulted in potent (partial) agonists (guinea pig right atrium, [35S]GTPγS and ß-arrestin2 recruitment assays) with human (h) H2R affinities in the one-digit nanomolar range (pKi (139, UR-KAT523): 8.35; pKi (157, UR-MB-69): 8.69). Most of the compounds presented here exhibited an excellent selectivity profile towards the hH2R, e.g. 157 being at least 3800-fold selective within the histamine receptor family. The structural similarities of our monomeric ligands to pramipexole (6), a dopamine receptor agonist, suggested an investigation of the binding behavior at those receptors. The target compounds were (partial) agonists with moderate affinity at the hD2longR and agonists with high affinity at the hD3R (e.g. pKi (139, UR-KAT523): 7.80; pKi (157, UR-MB-69): 8.06). In summary, we developed a series of novel, more drug-like H2R and D3R agonists for the application in recombinant systems in which either the H2R or the D3R is solely expressed. Furthermore, our ligands are promising lead compounds in the development of selective H2R agonists for future in vivo studies or experiments utilizing primary tissue to unravel the role and function of the H2R in the CNS.

Structural modifications in the distal, regulatory region of histamine H3 receptor antagonists leading to the identification of a potent anti-obesity agent.

A series of 4-pyridylpiperazine derivatives with varying regulatory region substituents proved to be potent histamine H3 receptor (H3R) ligands in the nanomolar concentration range. The most influential modification that affected the affinity toward the H3R appeared by introducing electron-withdrawing moieties into the distal aromatic ring. In order to finally discuss the influence of the characteristic 4-pyridylpiperazine moiety on H3R affinity, two Ciproxifan analogues 2 and 3 with a slight modification in their basic part were obtained. The replacement of piperazine in 3 with piperidine in compound 2, led to slightly reduced affinity towards the H3R (Ki = 3.17 and 7.70 nM, respectively). In fact, 3 showed the highest antagonistic properties among all compounds in this series, hence affirming our previous assumptions, that the 4-pyridylpiperazine moiety is the key element for suitable interaction with the human histamine H3 receptor. While its structural replacement to piperidine is also tolerated for H3R binding, the heteroaromatic 4-pyridyl moiety seems to be essential for proper ligand-receptor interaction. The putative protein-ligand interactions responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at the H3R, as well as drug-like properties of ligands were evaluated using in vitro methods. Moreover, pharmacological in vivo test results of compound 9 (structural analogue of Abbott's A-331440) clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound.

NanoBRET binding assay for histamine H2 receptor ligands using live recombinant HEK293T cells.

Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pKd = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pKi values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.