Phosphorylation-induced unfolding regulates p19(INK4d) during the human cell cycle

Cell cycle progression is tightly controlled in healthy organisms and often perturbed in human diseases, including, most prominently, many forms of cancers. Cyclin-dependent protein kinases and their inhibitors, such as p19(INK4d), regulate the different stages of the cell cycle. Here, we demonstrate how sequential phosphorylation of p19(INK4d) at two sites first destabilizes and then unfolds the N-terminal half of the protein, which dissociates its cyclin-dependent protein kinase-inhibitory complex and primes p19(INK4d) for cellular degradation. Our results define a structural mechanism by which phosphorylation-induced protein unfolding controls a key step in cell cycle progression

Small-molecule inhibitors of nisin resistance protein NSR from the human pathogen Streptococcus agalactiae

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules

N‑Terminal Phosphorylation of Parathyroid Hormone (PTH) Abolishes Its Receptor Activity

The parathyroid hormone (PTH) is an 84-residue peptide, which regulates the blood Ca²⁺ level via GPCR binding and subsequent activation of intracellular signaling cascades. PTH is posttranslationally phosphorylated in the parathyroid glands; however, the functional significance of this processes is not well characterized. In the present study, mass spectrometric analysis revealed three sites of phosphorylation, and NMR spectroscopy assigned Ser1, Ser3, and Ser17 as modified sites. These sites are located at the N-terminus of the hormone, which is important for receptor recognition and activation. NMR shows further that the three phosphate groups remotely disturb the α-helical propensity up to Ala36. An intracellular cAMP accumulation assay elucidated the biological significance of this phosphorylation because it ablated the PTH-mediated signaling. Our studies thus shed light on functional implications of phosphorylation at native PTH as an additional level of regulation

Time-resolved structural analysis of an RNA-cleaving DNA catalyst

The 10–23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10–23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme–RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes

Total Synthesis of the Antimycobacterial Natural Product Chlorflavonin and Analogs via a Late-Stage Ruthenium(II)-Catalyzed ortho-C(sp2)-H-Hydroxylation

The continuous, worldwide spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) endanger the World Health Organization’s (WHO) goal to end the global TB pandemic by the year 2035. During the past 50 years, very few new drugs have been approved by medical agencies to treat drug-resistant TB. Therefore, the development of novel antimycobacterial drug candidates to combat the threat of drug-resistant TB is urgent. In this work, we developed and optimized a total synthesis of the antimycobacterial natural flavonoid chlorflavonin by selective ruthenium(II)-catalyzed ortho-C(sp(2))-H-hydroxylation of a substituted 3′-methoxyflavonoid skeleton. We extended our methodology to synthesize a small compound library of 14 structural analogs. The new analogs were tested for their antimycobacterial in vitro activity against Mycobacterium tuberculosis (Mtb) and their cytotoxicity against various human cell lines. The most promising new analog bromflavonin exhibited improved antimycobacterial in vitro activity against the virulent H37Rv strain of Mtb (Minimal Inhibitory Concentrations (MIC(90)) = 0.78 μm). In addition, we determined the chemical and metabolic stability as well as the pK(a) values of chlorflavonin and bromflavonin. Furthermore, we established a quantitative structure–activity relationship model using a thermodynamic integration approach. Our computations may be used for suggesting further structural changes to develop improved derivatives

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is K(lig) = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML

Development of a First-in-Class Small Molecule Inhibitor of the C-terminal Hsp90 Dimerization

Heat shock protein 90 (Hsp90) is a promising therapeutic target due to its involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby contributing in exerting anti-apoptotic effects, which is essential for the malignant transformation and progression of several tumor types. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain (NTD) of Hsp90. However, adverse effects, including induction of the pro-survival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded clinical approval of these Hsp90i. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR and, thus, emerge as a promising alternative approach to target Hsp90. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with this essential dimerization process by small-molecule protein-protein interaction (PPI) inhibitors is a promising strategy for anticancer drug research. We have developed the first-in-class small molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitors (TKIs) resistant leukemia cells, without inducing HSR

Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization

Heat shock proteins 90 (Hsp90) are promising therapeutic targets due to their involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby exerting antiapoptotic effects, which is essential for the malignant transformation and tumor progression. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain of Hsp90. However, adverse effects, including induction of the prosurvival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded their clinical approval. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with the dimerization process by small-molecule protein–protein interaction inhibitors is a promising strategy for anticancer drug research. We have developed a first-in-class small-molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity, and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitor-resistant leukemia cells, without inducing HSR