In-Cell Testing of Zinc-Dependent Histone Deacetylase Inhibitors in the Presence of Class-Selective Fluorogenic Substrates: Potential and Limitations of the Method.

The development of anticancer drugs based on zinc-dependent histone deacetylase inhibitors (HDACi) has acquired great practical significance over the past decade. The most important HDACi characteristics are selectivity and strength of inhibition since they determine the mechanisms of therapeutic action. For in-cell testing of the selectivity of de novo-synthesized HDACi, Western blot analysis of the level of acetylation of bona fide protein substrates of HDACs of each class is usually used. However, the high labor intensity of this method prevents its widespread use in inhibitor screening. We developed an in-cell high-throughput screening method based on the use of three subtype-selective fluorogenic substrates of the general structure Boc-Lys(Acyl)-AMC, which in many cases makes it possible to determine the selectivity of HDACi at the class level. However, we found that the additional inhibitory activity of HDACi against metallo-ß-lactamase domain-containing protein 2 (MBLAC2) leads to testing errors.

Effects of natural polymorphisms in SARS-CoV-2 RNA-dependent RNA polymerase on its activity and sensitivity to inhibitors in vitro.

SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is the key enzyme required for viral replication and mRNA synthesis. RdRp is one of the most conserved viral proteins and a promising target for antiviral drugs and inhibitors. At the same time, analysis of public databases reveals multiple variants of SARS-CoV-2 genomes with substitutions in the catalytic RdRp subunit nsp12. Structural mapping of these mutations suggests that some of them may affect the interactions of nsp12 with its cofactors nsp7/nsp8 as well as with RNA substrates. We have obtained several mutations of these types and demonstrated that some of them decrease specific activity of RdRp in vitro, possibly by changing RdRp assembly and/or its interactions with RNA. Therefore, natural polymorphisms in RdRp may potentially affect viral replication. Furthermore, we have synthesized a series of polyphenol and diketoacid derivatives based on previously studied inhibitors of hepatitis C virus RdRp and found that several of them can inhibit SARS-CoV-2 RdRp. Tested mutations in RdRp do not have strong effects on the efficiency of inhibition. Further development of more efficient non-nucleoside inhibitors of SARS-CoV-2 RdRp should take into account the existence of multiple polymorphic variants of RdRp.

Selective Inhibition of HDAC Class I Sensitizes Leukemia and Neuroblastoma Cells to Anticancer Drugs.

The acquired resistance of neuroblastoma (NB) and leukemia cells to anticancer therapy remains the major challenge in the treatment of patients with these diseases. Although targeted therapy, such as receptor tyrosine kinase (RTK) inhibitors, has been introduced into clinical practice, its efficacy is limited to patients harboring mutant kinases. Through the analysis of transcriptomic data of 701 leukemia and NB patient samples and cell lines, we revealed that the expression of RTK, such as KIT, FLT3, AXL, FGFR3, and NTRK1, is linked with HDAC class I. Although HDAC inhibitors have antitumor activity, they also have high whole-body toxicity. We developed a novel belinostat derivative named hydrazostat, which targets HDAC class I with limited off-target effects. We compared the toxicity of these drugs within the panel of leukemia and NB cell lines. Next, we revealed that HDAC inhibition with hydrazostat reactivates NTRK1, FGFR3, ROR2, KIT, and FLT3 expression. Based on this finding, we tested the efficacy of hydrazostat in combination with RTK inhibitor imatinib. Additionally, we show the ability of hydrazostat to enhance venetoclax-induced apoptosis. Thus, we reveal the connection between HDACs and RTK and describe a useful strategy to overcome the complications of single-agent therapies.

Pre-Senescence Induction in Hepatoma Cells Favors Hepatitis C Virus Replication and Can Be Used in Exploring Antiviral Potential of Histone Deacetylase Inhibitors.

Recent evidence suggests that fibrotic liver injury in patients with chronic hepatitis C correlates with cellular senescence in damaged liver tissue. However, it is still unclear how senescence can affect replication of the hepatitis C virus (HCV). In this work, we report that an inhibitor of cyclin-dependent kinases 4/6, palbociclib, not only induced in hepatoma cells a pre-senescent cellular phenotype, including G1 arrest in the cell cycle, but also accelerated viral replicon multiplication. Importantly, suppression of HCV replication by direct acting antivirals (DAAs) was barely affected by pre-senescence induction, and vice versa, the antiviral activities of host-targeting agents (HTAs), such as inhibitors of human histone deacetylases (HDACi), produced a wide range of reactions-from a dramatic reduction to a noticeable increase. It is very likely that under conditions of the G1 arrest in the cell cycle, HDACi exhibit their actual antiviral potency, since their inherent anticancer activity that complicates the interpretation of test results is minimized.

Electron-Vibrational Spectra and Dynamics of the Lutein Molecule.

The carotenoid molecules such as lutein play an important role in the absorption of light and the following transfer of energy during photosynthesis. However, the study of these processes by the experimental methods only is quite difficult because some of the transitions between the electronic states of carotenoids are optically forbidden and the effect of vibrational states change also must be taken into account. In the present work, electronic-vibrational states of the lutein molecule in the LHCII complex of higher plants and in the diethyl ether solution were described using the ab initio methods. For lutein of LHCII, the electronic energy transfer processes were modeled. The role of the "hot" S1 states of lutein was shown to be of great importance.

Structural isomers of cinnamic hydroxamic acids block HCV replication via different mechanisms.

A set of ortho-, meta- and para-substituted cinnamic hydroxamic acids (CHAs) was synthesized. In each series of structural isomers, a phenyl substituent was linked to an aromatic ring of the parent cinnamic acid via a linker of one to four atoms in length. Using a cell test system with the full-length replicon of hepatitis C virus (HCV), we established a relationship between the suppression of HCV replicon propagation and the inhibition of class I/IIb histone deacetylases (HDACs). Anti-HCV activity correlated with the inhibition of HDAC8 in the case of ortho-CHAs, while in the case of meta-CHAs it correlated with the inhibition of HDAC1/2/3 and HDAC6. The antiviral activity of para-CHAs was many times stronger than that of meta-CHAs with about the same efficiency of HDAC1/2/3/6 inhibition, which indicated the existence of an additional cell target that does not belong to the studied group of HDACs.

Synthesis of N'-propylhydrazide analogs of hydroxamic inhibitors of histone deacetylases (HDACs) and evaluation of their impact on activities of HDACs and replication of hepatitis C virus (HCV).

N'-Propylhydrazide analogs of hydroxamic inhibitors of histone deacetylases (HDACs), including tubastatin A, vorinostat and belinostat, were synthesized. All prepared compounds inhibited HDAC1/2/3, but not HDAC6, except for one hydrazide analog of HDAC4/5/7 inhibitor that was completely inactive. A novel 4-substituted derivative of N'-propylbenzohydrazide with extremely high anti-HCV activity was discovered.

Rhodium(III) Complex with a Bulky Cyclopentadienyl Ligand as a Catalyst for Regioselective Synthesis of Dihydroisoquinolones through C-H Activation of Arylhydroxamic Acids.

Catalytic reaction of arylhydroxamic acids with alkenes represents a convenient method for preparation of biologically active dihydroisoquinolones. Here, the rhodium(III) complex [(C5 H2 tBu2 CH2 tBu)RhCl2 ]2 , which allows one to carry out such reactions with high regioselectivity to obtain 4-substituted dihydroisoquinolones in 72-97 % yields, is described. The regioselectivity is provided by the bulky cyclopentadienyl ligand of the catalyst, which is formed through a [2+2+1] cyclotrimerization of tert-butylacetylene. The catalytic reaction tolerates various distant functional groups in alkenes, but is inhibited by bulky (e.g., tBu) or strongly coordinating (e.g., imidazolyl) substituents. Some of the prepared dihydroisoquinolones effectively inhibit growth of phytopathogenic fungi.

Calculation of the excited states properties of LH1 complex of Thermochromatium tepidum.

Calculation of the excited states properties of pigment complexes is one of the key problems in the photosynthesis research. The excited states of LH1 complex of Thermochromatium tepidum were studied by means of the high-precision quantum chemistry methods. The influence of different parameters of the calculation procedure was examined. The optimal scheme of calculation was chosen by comparison of calculated results with the experimental data on absorption, electronic and magnetic circular dichroism spectra. The high importance of the account of the second excited states of bacteriochlorophylls and of site heterogeneity was shown. © 2018 Wiley Periodicals, Inc.

Pyridine hydroxamic acids are specific anti-HCV agents affecting HDAC6.

Recently we reported benzohydroxamic acids (BHAs) as potent and selective inhibitors of hepatitis C virus (HCV) replicon propagation. In this work 12 pyridine hydroxamic acids (PHAs) were synthesized and tested in full-genome replicon assay. It was found that PHAs possessed very similar anti-HCV properties compared to BHAs. Both classes of hydroxamic acids caused hyperacetylation of α-tubulin pointing to inhibition of histone deacetylase 6 (HDAC6) as part of their antiviral activity. The tested compounds did not inhibit the growth of poliovirus, displaying high selectivity against HCV.

Self-trapped leaky waves in lattices: discrete and Bragg soleakons.

We propose lattice soleakons: self-trapped waves that self-consistently populate slowly-attenuating leaky modes of their self-induced defects in periodic potentials. Two types, discrete and Bragg, lattice soleakons are predicted. Discrete soleakons that are supported by combination of self-focusing and self-defocusing nonlinearities propagate robustly for long propagation distances. They eventually abruptly disintegrate because they emit power to infinity at an increasing pace. In contrast, Bragg soleakons self-trap by only self-focusing nonlinearity. Also, they do not disintegrate because they emit power at a decreasing rate.

Benzohydroxamic acids as potent and selective anti-HCV agents.

A diverse collection of 40 derivatives of benzohydroxamic acid (BHAs) of various structural groups were synthesized and tested against hepatitis C virus (HCV) in full-genome replicon assay. Some of these compounds demonstrated an exceptional activity, suppressing viral replication at sub-micromolar concentrations. The compounds were inactive against key viral enzymes NS3, and NS5B in vitro assays, suggesting host cell inhibition target(s). The testing results were consistent with metal coordination by the BHAs hydroxamic group in complex with a target(s). Remarkably, this class of compounds did not suppress poliomyelitis virus (PV) propagation in RD cells indicating a specific antiviral activity of BHAs against HCV.

Finite element simulation of a perturbed axial-symmetric whispering-gallery mode and its use for intensity enhancement with a nanoparticle coupled to a microtoroid.

We present an optical mode solver for a whispering gallery resonator coupled to an adjacent arbitrary shaped nano-particle that breaks the axial symmetry of the resonator. Such a hybrid resonator-nanoparticle is similar to what was recently used for bio-detection and for field enhancement. We demonstrate our solver by parametrically studying a toroid-nanoplasmonic device and get the optimal nano-plasmonic size for maximal enhancement. We investigate cases near a plasmonic resonance as well as far from a plasmonic resonance. Unlike common plasmons that typically benefit from working near their resonance, here working far from plasmonic resonance provides comparable performance. This is because the plasmonic resonance enhancement is accompanied by cavity quality degradation through plasmonic absorption.

Reactive rifampicin derivative able to damage transcription complex.

Rifampicin (Rif) is powerful broad spectrum antibiotic that targets bacterial RNA polymerase (RNAP) by blocking the transcript exit channel. The performance of the drug can be further enhanced by tagging with active chemical groups that produce collateral damage. We explored this principle by tethering Rif to Fe(2+)-EDTA chelate. Modified drug retained high binding affinity to RNAP and caused localized cleavage of the enzyme and promoter DNA. Analysis of the degradation products revealed the cleavage of RNAP ß subunit at the sites involved in the drug binding, while DNA was selectively seized in the vicinity of the transcription start site. The synthesized Rif derivative exemplifies "aggressive" types of drugs that can be especially useful for TB treatment by attacking the nongrowing dormant form of the mycobacterium, which is hardly susceptible to "passive" drugs.

New cross-linking quinoline and quinolone derivatives for sensitive fluorescent labeling.

A variety of contemporary analytical platforms, utilized in technical and biological applications, take advantage of labeling the objects of interest with fluorescent tracers-compounds that can be easily and sensitively detected. Here we describe the synthesis of new fluorescent quinoline and quinolone compounds, whose light emission can be conveniently tuned by simple structural modifications. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. In addition, we also describe simple efficient derivatization reactions that allow introduction of amine- or click-reactive cross-linking groups into the fluorophores. The reactivity of synthesized compounds was confirmed in reactions with low molecular weight nucleophiles, or alkynes, as well as with click-reactive DNA-oligonucleotide containing synthetically introduced alkyne groups. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, living cells and small cellular metabolites. Obtained compounds are characterized using NMR, steady-state fluorescence spectroscopy as well as UV absorption spectroscopy.

Attosecond pulses with sophisticated spatio-spectral waveforms: spatio-spectral Airy and auto-focusing beams.

We propose a scheme for producing attosecond pulses with sophisticated spatio-spectral waveforms. The profile of a seed attosecond pulse is modified and its central frequency is up-converted through interaction with an infrared pump pulse. The transverse profile of the infrared beam and a spatiotemporal shift between the seed and infrared pulses are used for manipulating the spatio-spectral waveform of the generated pulse beam. We present several examples of sophisticated isolated attosecond pulse beam generation, including spatio-spectral Airy beam that exhibits prismatic self-bending effect and a beam undergoing auto-focusing to a sub-micron spot without the need of a focusing lens or nonlinearity.

Sawtooth grating-assisted phase-matching.

We show that a sawtooth phase-modulation is the optimal profile for grating assisted phase matching (GAPM). Perfect (sharp) sawtooth modulation fully corrects the phase-mismatch, exhibiting conversion equal to conventional phase matching, while smoothened, approximate sawtooth structures are more efficient than sinusoidal or square GAPM modulations that were previously studied. As an example, we demonstrate numerically optically-induced sawtooth GAPM for high harmonic generation. Sawtooth GAPM is the most efficient method for increasing the conversion efficiency of high harmonic generation through quasi-phase-matching, with an ultimate efficiency that closely matches the ideal phase-matching case.

Luminescent probes for ultrasensitive detection of nucleic acids.

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.

Homogeneous fluorescent assay for RNA polymerase.

A new method for determination of RNA polymerase (RNAP) activity is presented. The method uses nucleoside tri- and tetraphosphate derivatives carrying 4-methylumbelliferone residue at the terminal phosphate. Incorporation of such compounds in RNA by RNA polymerase is accompanied by release of di- and triphosphate derivatives of 4-methylumbelliferone. Subsequent treatment by alkaline phosphatase produces free 4-methylumbelliferone that is highly fluorescent and can be easily detected. The sensitivity of the method is higher than that reported in previous studies. The validity of the assay has been demonstrated by retrieving the RNAP inhibitors from a collection of 16,000 compounds.