Covalent-reversible peptide-based protease inhibitors. Design, synthesis, and clinical success stories.

Dysregulated human peptidases are implicated in a large variety of diseases such as cancer, hypertension, and neurodegeneration. Viral proteases for their part are crucial for the pathogens' maturation and assembly. Several decades of research were devoted to exploring these precious therapeutic targets, often addressing them with synthetic substrate-based inhibitors to elucidate their biological roles and develop medications. The rational design of peptide-based inhibitors offered a rapid pathway to obtain a variety of research tools and drug candidates. Non-covalent modifiers were historically the first choice for protease inhibition due to their reversible enzyme binding mode and thus presumably safer profile. However, in recent years, covalent-irreversible inhibitors are having a resurgence with dramatic increase of their related publications, preclinical and clinical trials, and FDA-approved drugs. Depending on the context, covalent modifiers could provide more effective and selective drug candidates, hence requiring lower doses, thereby limiting off-target effects. Additionally, such molecules seem more suitable to tackle the crucial issue of cancer and viral drug resistances. At the frontier of reversible and irreversible based inhibitors, a new drug class, the covalent-reversible peptide-based inhibitors, has emerged with the FDA approval of Bortezomib in 2003, shortly followed by 4 other listings to date. The highlight in the field is the breathtakingly fast development of the first oral COVID-19 medication, Nirmatrelvir. Covalent-reversible inhibitors can hipothetically provide the safety of the reversible modifiers combined with the high potency and specificity of their irreversible counterparts. Herein, we will present the main groups of covalent-reversible peptide-based inhibitors, focusing on their design, synthesis, and successful drug development programs.

Diversity of mitochondrial DNA in 3 species of great whales before and after modern whaling.

The 20th century commercial whaling industry severely reduced populations of great whales throughout the Southern Hemisphere. The effect of this exploitation on genetic diversity and population structure remains largely undescribed. Here, we compare pre- and post-whaling diversity of mitochondrial DNA (mtDNA) control region sequences for 3 great whales in the South Atlantic, such as the blue, humpback, and fin whale. Pre-whaling diversity is described from mtDNA extracted from bones collected near abandoned whaling stations, primarily from the South Atlantic island of South Georgia. These bones are known to represent the first stage of 20th century whaling and thus pre-whaling diversity of these populations. Post-whaling diversity is described from previously published studies reporting large-scale sampling of living whales in the Southern Hemisphere. Despite relatively high levels of surviving genetic diversity in the post-whaling populations, we found evidence of a probable loss of mtDNA lineages in all 3 species. This is evidenced by the detection of a large number of haplotypes found in the pre-whaling samples that are not present in the post-whaling samples. A rarefaction analysis further supports a loss of haplotypes in the South Atlantic humpback and Antarctic blue whale populations. The bones from former whaling stations in the South Atlantic represent a remarkable molecular archive for further investigation of the decline and ongoing recovery in the great whales of the Southern Hemisphere.

The Unexpected Helical Supramolecular Assembly of a Simple Achiral Acetamide Tecton Generates Selective Water Channels.

Invited for the cover of this issue is the collaborative research team coordinated by Arie van der Lee at the University of Montpellier. The image depicts chiral channels with highly mobile water molecules resulting from the robust self-organization of a simple achiral acetamide. Fully reversible release and re-uptake of water molecules takes place near ambient conditions, with efficient water transport and a good selectivity against NaCl suggesting it to be an efficient candidate for desalination processes. Read the full text of the article at 10.1002/chem.20200383.

The Unexpected Helical Supramolecular Assembly of a Simple Achiral Acetamide Tecton Generates Selective Water Channels.

Achiral 2-hydroxy-N-(diphenylmethyl)acetamide (HNDPA) crystallizes in the P61 chiral space group as a hydrate, building up permeable chiral crystalline helical water channels. The crystallization-driven chiral self-resolution process is highly robust, with the same air-stable crystalline form readily obtained under a variety of conditions. Interestingly, the HNDPA supramolecular helix inner pore is filled by a helical water wire. The whole edifice is mainly stabilized by robust hydrogen bonds involving the HNDPA amide bonds and CH… π interactions between the HNDPA phenyl groups. The crystalline structure shows breathing behavior, with completely reversible release and re-uptake of water inside the chiral channel under ambient conditions. Importantly, the HNDPA channel is able to transport water very efficiently and selectively under biomimetic conditions. With a permeability per channel of 3.3 million water molecules per second in large unilamellar vesicles (LUV) and total selectivity against NaCl, the HNDPA channel is a very promising functional nanomaterial for future applications.

Development of highly sensitive fluorescent probes for the detection of ß-galactosidase activity - application to the real-time monitoring of senescence in live cells.

We report the development of four novel fluorescent probes to monitor the activity of the ß-galactosidase enzyme (ß-gal), in vitro and in living cells. The fluorophores are based on a 6-amino-styryl-benzothiazole push-pull core and display a strong ICT emission. The probes encompass the fluorescent motif that is connected to a ß-d-galactopyranoside moiety through a self-immolative benzyl carbamate linker (ßGal-1-4). The screening of four different fluorophores enabled us to access new light-up and two-band ratiometric reporters. The four probes, ßGal-1-4, exhibited an extremely fast response and over 200-fold fluorescence enhancement (ßGal-1) following the enzymatic cleavage of the ß-d-galactopyranoside unit. This rapid and extremely sensitive response allowed the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity; a widely used biomarker of senescence. More importantly, ßGal-1 also enabled us to monitor, in real-time, the emergence of senescence in live cells, i.e. the phenotypic transformation from normal to senescent cell. These findings underpin the fact that ßGal-1 may find useful applications in biomedical research. Importantly, ßGal-1 is suitable for epifluorescence and confocal microscopies, and flow cytometry techniques, which are among the most common analytical tools in biology.

Metastatic Melanoma: Insights Into the Evolution of the Treatments and Future Challenges.

Melanoma is the deadliest form of skin cancer. While associated survival prognosis is good when diagnosed early, it dramatically drops when melanoma progresses into its metastatic form. Prior to 2011, the favored therapies include interleukin-2 and chemotherapies, regardless of their low efficiency and their toxicity. Following key biological findings, two new types of therapy have been approved. First, there are the targeted therapies, which rely on small molecule B-Raf and MEK inhibitors and allow the treatment of patients with B-Raf mutated melanoma. Second, there are the immunotherapies, with anti-CTLA-4 and anti-PD-1 antibodies that are used for patients harboring a B-Raf wild-type status. Both approaches have significantly improved patient survival, compared with alkylating agents, in the treatment of unresectable melanoma. Herein, we review the evolution of the treatment of melanoma starting from early discoveries to current therapies. A focus will be provided on drug discovery, synthesis, and mode of action of relevant drugs and the future directions of the domain to overcome the emergence of the resistance events.

Assessment of new triplet forming artificial nucleobases as RNA ligands directed towards HCV IRES IIId loop.

We report the synthesis of two new artificial nucleobase scaffolds, 1 and 2, featuring adequate hydrogen bonding donors and acceptors for the molecular recognition of U:A and C:G base pairs, respectively. The tethering of these structures to various amino acids and the assessment of these artificial nucleobase-amino acid conjugates as RNA ligands against a model of HCV IRES IIId domain are also reported. Compound 1e displayed the highest affinity (Kd twice lower than neomycin - control). Moreover, it appears that this interaction is enthalpically and entropically favored.

On the Mechanism of the Digold(I)-Hydroxide-Catalysed Hydrophenoxylation of Alkynes.

Herein, we present a detailed investigation of the mechanistic aspects of the dual gold-catalysed hydrophenoxylation of alkynes by both experimental and computational methods. The dissociation of [{Au(NHC)}2 (µ-OH)][BF4 ] is essential to enter the catalytic cycle, and this step is favoured by the presence of bulky, non-coordinating counter ions. Moreover, in silico studies confirmed that phenol does not only act as a reactant, but also as a co-catalyst, lowering the energy barriers of several transition states. A gem-diaurated species might form during the reaction, but this lies deep within a potential energy well, and is likely to be an "off-cycle" rather than an "in-cycle" intermediate.

Scope and limitations of the dual-gold-catalysed hydrophenoxylation of alkynes.

Due to the synthetic advantages presented by the dual-gold-catalysed hydrophenoxylation of alkynes, a thorough study of this reaction was carried out in order to fully define the scope and limitations of the methodology. The protocol tolerates a wide range of functional groups, such as nitriles, ketones, esters, aldehydes, ketals, naphthyls, allyls or polyphenols, in a milder and more efficient manner than the previously reported methodologies. We have also identified that while we are able to use highly steric hindered phenols, small changes on the steric bulk of the alkynes have a dramatic effect on the reactivity. More importantly, we have observed that the use of substrates that facilitate the formation of diaurated species such as gem-diaurated or σ,π-digold-acetylide species, hinder the catalytic activity. Moreover, we have identified that the use of directing groups in unsymmetrical alkynes can help to achieve high regioselectivity in the hydrophenoxylation.

Direct synthesis of partially modified 2'-O-pivaloyloxymethyl RNAs by a base-labile protecting group strategy and their potential for prodrug-based gene-silencing applications.

An original and straightforward synthesis of partially modified 2'-O-pivaloyloxymethyl-substituted (PivOM-substituted) oligoribonucleotides has been achieved. The aim of this 2'-enzymolabile modification was to enhance nuclease stability of RNA and transmembrane transport. To make these modified RNAs easily available we developed a base-labile protecting group strategy with standard protections for nucleobases (acyl) and phosphates (cyanoethyl), a Q-linker and two different acetalester protection groups for 2'-OH: propionyloxymethyl (PrOM) and PivOM. Interestingly, orthogonal deprotection conditions based on anhydrous butylamine in THF were found to remove propionyloxymethyl groups selectively, while preserving PivOM groups. Duplex stability, circular dichroism studies and nuclease resistance, as well as the ability to inhibit gene expression of modified 2'-O-PivOM RNA, were evaluated.

[Au]/[Pd] Multicatalytic processes: direct one-pot access to benzo[c]chromenes and benzo[b]furans.

A new synthetic protocol that combines the advantages offered by eco-friendly solvent-free reactions and sequential transformations is reported. This strategy offers straightforward access to benzo[c]chromenes and benzo[b]furans from commercially available starting materials. This two-step, one-pot strategy consists of an Au-catalyzed hydrophenoxylation process followed by Pd-catalyzed C-H activation or Mizoroki-Heck reactions. The selectivity of the process towards C-H activation or Mizoroki-Heck reaction can be easily tuned.

Boron and nucleic acid chemistries: merging the best of both worlds.

At the intersection of nucleic acid and boron chemistries lies a thriving world of possibilities. During the past decades, the merging of these research domains has led to fascinating discoveries in different fields ranging from material to medical sciences. In recent years the interplay of these two worlds has gained a lot of attention, as can be judged by the increasing number of articles in which boron and nucleic acids stand out for their potential medicinal, biotechnological or analytical applications. In this review, we present an outline of this crossroads by focusing on both the interaction of boronated compounds with nucleic acids and the modification of nucleic acids by boron containing moieties.

Targeting a lineage-specific PI3KÉ£-Akt signaling module in acute myeloid leukemia using a heterobifunctional degrader molecule.

Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in nonmalignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue-restricted and/or tumor-restricted expression. In line with another recent report, we show here that, in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG/p110γ-PIK3R5/p101 axis inhibits protein kinase B/Akt signaling and compromises AML cell fitness. Furthermore, silencing the genes encoding PIK3CG/p110γ or PIK3R5/p101 sensitizes AML cells to established AML therapies. Importantly, we find that existing small-molecule inhibitors against PIK3CG are insufficient to achieve a sustained long-term antileukemic effect. To address this concern, we developed a proteolysis-targeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary samples from patients with AML and syngeneic mouse models.

MCB-613 exploits a collateral sensitivity in drug resistant EGFR-mutant non-small cell lung cancer through covalent inhibition of KEAP1.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR-mutant NSCLC cells.

Borononucleotides as substrates/binders for human NMP kinases: enzymatic and spectroscopic evaluation.

Borononucleotides are a family of natural nucleotide monophosphate analogues with a 5'-boronic acid function. As B-O-P linkages are known to be unstable in solution, we evaluated the ability of borononucleotides to be recognized by nucleoside monophosphate kinases and eventually foil the phosphorylation process. In this context, and with the idea of probing the influence of their size, shape, and flexibility, a library of borononucleotides were synthetized starting from the borononucleotide analogue of thymidine, which was shown to behave as a slow substrate of human TMP kinase. This study thus constitutes a good starting point for the development of new monophosphate mimics as potential substrates or ligands for NMP kinases.

Molecular cytogenetic characterization of the Amazon River dolphin Inia geoffrensis.

Classical and molecular cytogenetic (18S rDNA, telomeric sequence, and LINE-1 retrotransposon probes) studies were carried out to contribute to an understanding of the organization of repeated DNA elements in the Amazon River dolphin (boto, Inia geoffrensis). Twenty-seven specimens were examined, each presenting 2n = 44 chromosomes, the karyotype formula 12m + 14sm + 6st + 10t + XX/XY, and fundamental number (FN) = 74. C-positive heterochromatin was observed in terminal and interstitial positions, with the occurrence of polymorphism. Interstitial telomeric sequences were not observed. The nucleolar organizer region (NOR) was located at a single site on a smallest autosomal pair. LINE-1 was preferentially distributed in the euchromatin regions, with the greatest accumulation on the X chromosome. Although the karyotype structure in cetaceans is considered to be conserved, the boto karyotype demonstrated significant variations in its formula, heterochromatin distribution, and the location of the NOR compared to other cetacean species. These results contribute to knowledge of the chromosome organization in boto and to a better understanding of karyoevolution in cetaceans.

Extending the utility of [Pd(NHC)(cinnamyl)Cl] precatalysts: Direct arylation of heterocycles.

The use of [Pd(NHC)(cinnamyl)Cl] precatalysts in the direct arylation of heterocycles has been investigated. Among four different precatalysts, [Pd(SIPr)(cinnamyl)Cl] proved to be the most efficient promoter of the reaction. The C-H functionalization of sulfur- or nitrogen-containing heterocycles has been achieved at low catalyst loadings. These catalyst charges range from 0.1 to 0.01 mol % palladium.

Radiographic Outcomes following K-Wire Augmentation of Dorsal Spanning Plate Fixation for Intra-Articular Fractures of the Distal Radius.

Background Restoration of articular surface alignment is critical in treating intra-articular distal radius fractures. Dorsal spanning plate fixation functions as an internal distraction mechanism and can be advantageous in the setting of highly comminuted fracture patterns, polytrauma patients, and patients with radiocarpal instability. The addition of K-wires to support articular surface reduction potentially augments fracture repair stability. Questions/Purposes We examined the radiographic outcomes and maintenance of reduction in patients with comminuted intra-articular distal radius fractures treated with K-wire fixation of articular fragments followed by dorsal spanning plate application. Patients and Methods We reviewed 35 consecutive patients with complex intra-articular distal radius fractures treated with dorsal spanning plate and K-wire fixation between April 2016 and October 2019. AO classification was recorded: B1 (3), B3 (2), C2 (2), C3 (28). A two-tailed paired t -test was used to compare findings immediately post-dorsal spanning plate surgery and at final follow-up after dorsal spanning plate removal. Results Mean patient age was 43.3 years (19-78 years). Mean follow-up was 7.8 months (SD 4.3 months) from surgery and 2.5 months from pin removal (SD 2.6 months). All patients achieved radiographic union. Radial height (mean interval change (MIC) 0.2 mm, SD 2.2, p = 0.63), articular step-off (MIC 0.1 mm, SD 0.6 mm, p = 0.88), and radial inclination (MIC -1.1 degrees, SD 3.7 degrees, p = 0.10) did not change from post-surgery to final follow-up. Ulnar variance (MIC -0.9 mm, SD 2.0 mm, p = 0.02) and volar tilt (MIC -1.5 degrees, SD 4.4 degrees, p = 0.05) were found to have decreased. Conclusion Dorsal spanning plate augmented with K-wire fixation for comminuted intra-articular distal radius fractures in polytrauma patients allows for immediate weightbearing and maintains articular surface alignment at radiographic union and may provide better articular restoration than treatment with dorsal spanning plate alone. Level of Evidence This is a Level IV , therapeutic study.

Ultrasound-assisted one-pot three-component synthesis of new isoxazolines bearing sulfonamides and their evaluation against hematological malignancies.

In the present study, following a one-pot two-step protocol, we have synthesized novel sulfonamides-isoxazolines hybrids (3a-r) via a highly regioselective 1,3-dipolar cycloaddition. The present methodology capitalized on trichloroisocyanuric acid (TCCA) as a safe and ecological oxidant and chlorinating agent for the in-situ conversion of aldehydes to nitrile oxides in the presence of hydroxylamine hydrochloride, under ultrasound activation. These nitrile oxides could be engaged in 1,3-dipolar cycloaddition reactions with various alkene to afford the targeted sulfonamides-isoxazolines hybrids (3a-r). The latter were assessed for their antineoplastic activity against model leukemia cell lines (Chronic Myeloid Leukemia, K562 and Promyelocytic Leukemia, HL-60).

Resorbable Pins to Enhance Scaffold Retention in a Porcine Chondral Defect Model.

OBJECTIVE: Cartilage repair strategies have seen improvement in recent years, especially with the use of scaffolds that serve as a template for cartilage formation. However, current fixation strategies are inconsistent with regards to retention, may be technically challenging, or may damage adjacent tissues or the implant itself. Therefore, the goal of this study was to evaluate the retention and repair potential of cartilage scaffolds fixed with an easy-to-implement bioresorbable pin. DESIGN: Electrospun hyaluronic acid scaffolds were implanted into trochlear groove defects in 3 juvenile and 3 adult pigs to evaluate short-term retention (2 weeks; pin fixation vs. press-fit and fibrin fixation) and long-term repair (8 months; scaffold vs. microfracture), respectively. RESULTS: For the retention study, press-fit and fibrin fixation resulted in short-term scaffold dislodgment (n = 2 each), whereas pin fixation retained all scaffolds that were implanted (n = 6). Pin fixation did not cause any damage to the opposing patellar surface, and only minor changes in the subchondral bone were observed. For long-term repair, no differences were observed between microfracture and scaffold groups, in terms of second-look arthroscopy and indentation testing. On closer visualization with micro computed tomography and histology, a high degree of variability was observed between animals with regard to subchondral bone changes and cartilage repair quality, yet each Scaffold repair displayed similar properties to its matched microfracture control. CONCLUSIONS: In this study, pin fixation did not cause adverse events in either the short- or the long-term relative to controls, indicating that pin fixation successfully retained scaffolds within defects without inhibiting repair.