Design principles governing the development of theranostic anticancer agents and their nanoformulations with photoacoustic properties

The unmet need to develop novel approaches for cancer diagnosis and treatment has led to the evolution of theranostic agents, which usually include, in addition to the anticancer drug, an imaging agent based mostly on fluorescent agents. Over the past few years, a non-invasive photoacoustic imaging modality has been effectively integrated into theranostic agents. Herein, we shed light on the design principles governing the development of theranostic agents with photoacoustic properties, which can be formulated into nanocarriers to enhance their potency. Specifically, we provide an extensive analysis of their individual constituents including the imaging dyes, drugs, linkers, targeting moieties, and their formulation into nanocarriers. Along these lines, we present numerous relevant paradigms. Finally, we discuss the clinical relevance of the specific strategy, as also the limitations and future perspectives, and through this review, we envisage paving the way for the development of theranostic agents endowed with photoacoustic properties as effective anticancer medicines

Electronic sculpting of ligand-GPCR subtype selectivity:the case of angiotensin II

GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (<i>K</i>_i = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range

Multiple segmental and selective isotope labeling of large RNA for NMR structural studies

Multiple segmental and selective isotope labeling of RNA with three segments has been demonstrated by introducing an RNA segment, selectively labeled with 13C9/15N2/2H(1′, 3′, 4′, 5′, 5′′)-labeled uridine residues, into the central position of the 20 kDa ε-RNA of Duck Hepatitis B Virus. The RNA molecules were produced via two efficient protocols: a two-step protocol, which uses T4 DNA ligase and T4 RNA ligase 1, and a one-pot protocol, which uses T4 RNA ligase 1 alone. With T4 RNA ligase 1 all not-to-be-ligated termini are usually protected to prevent formation of side products. We show that such labor-intensive protection of termini is not required, provided segmentation sites can be chosen such that the segments fold into the target structure or target-like structures and thus are not trapped into stable alternate structures. These sites can be reliably predicted via DINAMelt. The simplified NMR spectrum provided evidence for the presence of a U28 H3-imino resonance, previously obscured in the fully labeled sample, and thus of the non-canonical base pair U28:C37. The demonstrated multiple segmental labeling protocols are generally applicable to large RNA molecules and can be extended to more than three segments

Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tetheredligand

Thrombin is the most potent agonist of human platelets and its effects are primarily mediated through the protease-activated receptors (PARs)-1 and -4. Although PAR-1 has higher affinity for thrombin than PAR-4, both receptors contribute to thrombin-mediated actions on platelets. Recently, a potent and selective PAR-1 antagonist (vorapaxar) was approved for clinical use in selected patients. In contrast, despite the fact that several PAR-4 antagonists have been developed, few of them have been tested in clinical trials. The aim of the present study was to elucidate the molecular requirements involving the PAR-4 mechanism of activation by peptide analogues of its tethered-ligand. Eight synthetic PAR-4 tethered-ligand peptide analogues were synthesized and studied for their agonistic/antagonistic potency and selectivity toward human washed platelet aggregation, using light transmittance aggregometry. In addition, in silico studies were conducted to describe the receptor–peptide interactions that are developed following PAR-4 exposure to the above analogues. To provide a first structure-activity relationship rationale on the bioactivity profiles recorded for the studied analogues, molecular docking was applied in a homology model of PAR-4, derived using the crystal structure of PAR-1. The following peptide analogues were synthesized: AYPGKF-NH2 (1), GYPGKF-NH2 (2), AcAYPGKF-NH2 (3), trans-cinnamoyl-AYPGKF-NH2 (4), YPGKF-NH2 (5), Ac-YPGKF-NH2 (6), transcinnamoyl-YPGKF-NH2 (7), and caffeoyl-YPGKF-NH2 (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC50 value of 26.2 μM. Substitution of Ala-1 with Gly-1 resulted in peptide (2), which significantly reduces the agonistic potency of peptide (1) by 25- fold. Importantly, substitution of Ala-1 with trans-cinnamoyl-1 resulted in peptide (7), which completely abolishes the agonistic activity of peptide (1) and renders it with a potent antagonistic activity toward peptide (1)-induced platelet aggregation. All other peptides tested were inactive. Tyr-2, residue, along with its neighboring environment was a key determinant in the PAR-4 recognition mode. When the neighboring residues to Tyr-2 provided an optimum spatial ability for the ligand to enter into the binding site of the transmembrane receptor, a biological response was propagated. These results were compared with the predicted binding poses of small molecule antagonists of PAR-4, denoted as YD-3, ML-354, and BMS-986120. π–π stacking interaction with Tyr-183 appears to be critical and common for both small molecules antagonists and the peptide trans-cinnamoyl-YPGKFNH2. Conclusively, the lipophilicity, size, and aromatic nature of the residue preceding Tyr-2 are determining factors on whether a human platelet PAR-4 tethered-ligand peptide analogue will exert an agonistic or antagonistic activit

Exploring the interactions of irbesartan and irbesartan–2-hydroxypropyl-β-cyclodextrin complex with model membranes

The interactions of irbesartan (IRB) and irbesartan–2-hydroxypropyl-β-cyclodextrin (HP-β-CD) complex with Dipalmitoyl Phosphatidylcholine (DPPC) bilayers have been explored utilizing an array of biophysical techniques ranging from Differential Scanning Calorimetry (DSC), Small angle X-ray Scattering (SAXS), ESI Mass-Spectrometry (ESI-MS) and solid state Nuclear Magnetic Resonance (ssNMR). Molecular Dynamics (MD) calculations have been also conducted to complement the experimental results. Irbesartan was found to be embedded in the lipid membrane core and to affect the phase transition properties of the DPPC bilayers. SAXS studies revealed that irbesartan alone does not display perfect solvation since some coexisting irbesartan crystallites are present. In its complexed form IRB gets fully solvated in the membranes showing that encapsulation of IRB in HP-β-CD may have beneficial effects in the ADME properties of this drug. MD experiments revealed the topological and orientational integration of irbesartan into the phospholipid bilayer being placed at about 1 nm from the membrane centre

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental 13C/15N/2H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2′ of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively 13C9/15N3/2H(1′,2″,3′,4′,5′,5″)-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively 13C9/15N3/2H(1′,2″,3′,4′,5′,5″)-dC and 13C9/15N2/2H(1′,2″,3′,4′,5′,5″)-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective 2H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1′–H2′ and C2′–H2′ resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments

Molecular Biomechanics: The Molecular Basis of How Forces Regulate Cellular Function

Recent advances have led to the emergence of molecular biomechanics as an essential element of modern biology. These efforts focus on theoretical and experimental studies of the mechanics of proteins and nucleic acids, and the understanding of the molecular mechanisms of stress transmission, mechanosensing and mechanotransduction in living cells. In particular, single-molecule biomechanics studies of proteins and DNA, and mechanochemical coupling in biomolecular motors have demonstrated the critical importance of molecular mechanics as a new frontier in bioengineering and life sciences. To stimulate a more systematic study of the basic issues in molecular biomechanics, and attract a broader range of researchers to enter this emerging field, here we discuss its significance and relevance, describe the important issues to be addressed and the most critical questions to be answered, summarize both experimental and theoretical/computational challenges, and identify some short-term and long-term goals for the field. The needs to train young researchers in molecular biomechanics with a broader knowledge base, and to bridge and integrate molecular, subcellular and cellular level studies of biomechanics are articulated.National Institutes of Health (U.S.) (grant UO1HL80711-05 to GB)National Institutes of Health (U.S.) (grant R01GM076689-01)National Institutes of Health (U.S.) (grant R01AR033236-26)National Institutes of Health (U.S.) (grant R01GM087677-01A1)National Institutes of Health (U.S.) (grant R01AI44902)National Institutes of Health (U.S.) (grant R01AI38282)National Science Foundation (U.S.) (grant CMMI-0645054)National Science Foundation (U.S.) (grant CBET-0829205)National Science Foundation (U.S.) (grant CAREER-0955291

Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron–electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to ‘knock out’ superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies

Ligand–Receptor Interactions and Drug Design

In silico rational drug design is one of the major pylons in the drug discovery process. Drugs usually act on specific targets such as proteins, DNA, and lipid bilayers. Thus, molecular docking is an essential part of the rational drug design process. Molecular docking uses specific algorithms and scoring functions to reveal the strength of the interaction of the ligand to its target. AutoDock is a molecular docking suite that offers a variety of algorithms to tackle specific problems. These algorithms include Monte Carlo Simulated Annealing (SA), a Genetic Algorithm (GA), and a hybrid local search GA, also known as the Lamarckian Genetic Algorithm (LGA). This chapter aims to acquaint the reader with the docking process using AutoDockTools (GUI of AutoDock). Furthermore, herein is described the docking process of calf thymus DNA with three metal complexes, as a potential metallo-therapeutics as also the docking process of the plant flavonoid quercetin to the antiapoptotic protein BcL-xL. © 2021, Springer Science+Business Media, LLC, part of Springer Nature