Identification of highly potent and selective HTRA1 inhibitors.

High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure-activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50's less than 15 nM and excellent selectivity following a screen of 35 proteases.

Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.

Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

The cumulative incidence and risk factors associated with 5-year conversion to knee arthroplasty following primary meniscus repair or primary meniscectomy.

Background: This study aimed to observe the 5-year knee arthroplasty conversion incidence rate and associated risk factors in patients who underwent meniscus procedures. Methods: Using a national database, we analyzed patients who had undergone primary meniscus repair or meniscectomy without prior knee surgeries. The cumulative knee arthroplasty conversion incidence was determined via Kaplan Meier analysis. Risk factors for conversion within 5 years were assessed using a Cox proportional hazard ratio model, with results as hazard ratios (HR). Results: 8125 patients had meniscus repair, while 240,209 had meniscectomy. 5-year conversion rates: repair 1.7%, meniscectomy 8.4%. Arthroplasty likelihood decreased as age decreased for repair (70+ [HR: 162.20]; 60-69 [HR: 81.64]; 50-59 [HR: 49.85]; 40-49 [HR: 17.79]; p < 0.001 all). Additional risk factors included male sex (HR: 0.35; p < 0.001) and higher Charlson Comorbidity Index (CCI) (CCI1 [HR: 1.28; p = 0.012]). For meniscectomy, arthroplasty likelihood also decreased with age (70+ [HR: 99.41]; 60-69 [HR: 84.57]; 50-59 [HR: 66.60]; 40-49 [HR: 36.15]; 30-39 [HR: 10.18]; p < 0.001 all). Additional risk factors included male sex (HR: 0.68; p < 0.001), obesity (HR: 1.18; p < 0.001), smoking (HR: 0.1.12; p = 0.010), and higher CCI (CCI1 [HR: 1.25]; CCI2 [HR 1.39]; CCI3+ [HR 1.46]; p < 0.001 all). Conclusion: This study revealed the national 5-year conversion incidence following primary meniscus repair (1.7%) and meniscectomy (8.4%). It also enhanced understanding of age, sex, obesity, smoking, comorbidities (CCI), and knee arthroplasty likelihood after meniscus procedures.

Antibody-mediated prevention of vaginal HIV transmission is dictated by IgG subclass in humanized mice.

HIV broadly neutralizing antibodies (bNAbs) are capable of both blocking viral entry and driving innate immune responses against HIV-infected cells through their Fc region. Vaccination or productive infection results in a polyclonal mixture of class-switched immunoglobulin G (IgG) antibodies composed of four subclasses, each encoding distinct Fc regions that differentially engage innate immune functions. Despite evidence that innate immunity contributes to protection, the relative contribution of individual IgG subclasses is unknown. Here, we used vectored immunoprophylaxis in humanized mice to interrogate the efficacy of individual IgG subclasses during prevention of vaginal HIV transmission by VRC07, a potent CD4-binding site-directed bNAb. We find that VRC07 IgG2, which lacks Fc-mediated functionality, exhibited substantially reduced protection in vivo relative to other subclasses. Low concentrations of highly functional VRC07 IgG1 yielded substantial protection against vaginal challenge, suggesting that interventions capable of eliciting modest titers of functional IgG subclasses may provide meaningful benefit against infection.

Multi-modal profiling of human fetal liver hematopoietic stem cells reveals the molecular signature of engraftment.

The human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression reveals a generalizable signature defining functional fetal liver hematopoietic stem cells and allows for the stratification of enrichment strategies with high translational potential. More precisely, our integrated analysis identifies CD201 (endothelial protein C receptor (EPCR), encoded by PROCR) as a marker that can specifically enrich for engraftment potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers. As such, it serves as a useful resource for the field and forms the basis for further biological exploration of strategies to retain the engraftment potential of hematopoietic stem cells ex vivo or induce this potential during in vitro hematopoietic stem cell generation.

Structure-based phylogeny identifies avoralstat as a TMPRSS2 inhibitor that prevents SARS-CoV-2 infection in mice.

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS coronavirus 2 (SARS-CoV-2) viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, avoralstat, PCI-27483, antipain, and soybean trypsin inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, avoralstat significantly reduced lung tissue titers and mitigated weight loss when administered prophylactically to mice susceptible to SARS-CoV-2, indicating its potential to be repositioned for coronavirus disease 2019 (COVID-19) prophylaxis in humans.

Peptidomimetics Therapeutics for Retinal Disease.

Ocular disorders originating in the retina can result in a partial or total loss of vision, making drug delivery to the retina of vital importance. However, effectively delivering drugs to the retina remains a challenge for ophthalmologists due to various anatomical and physicochemical barriers in the eye. This review introduces diverse administration routes and the accordant pharmacokinetic profiles of ocular drugs to aid in the development of safe and efficient drug delivery systems to the retina with a focus on peptidomimetics as a growing class of retinal drugs, which have great therapeutic potential and a high degree of specificity. We also discuss the pharmacokinetic profiles of small molecule drugs due to their structural similarity to small peptidomimetics. Lastly, various formulation strategies are suggested to overcome pharmacokinetic hurdles such as solubility, retention time, enzymatic degradation, tissue targeting, and membrane permeability. This knowledge can be used to help design ocular delivery platforms for peptidomimetics, not only for the treatment of various retinal diseases, but also for the selection of potential peptidomimetic drug targets.

Noncanonical Cation-π Cyclizations of Alkylidene ß-Ketoesters: Synthesis of Spiro-fused and Bridged Bicyclic Ring Systems.

Three cation-π cyclization cascades initiated at alkylidene ß-ketoesters bearing pendent alkenes are described. Depending upon the alkene substitution pattern and the reaction conditions employed, it is possible to achieve selective synthesis of the three different types of products, including 1-halo-3-carbomethoxycyclohexanes, spiro-fused tricyclic systems, and [4.3.1] bridged bicyclic ring systems. All three reactions begin with 6- endo addition of an olefin to the alkylidene ß-ketoester electrophile, followed by one of three different cation capture events.

Two cadmium coordination polymers with bridging acetate and phenyl-enedi-amine ligands that exhibit two-dimensional layered structures.

Poly[tetra-µ2-acetato-κ8O:O'-bis-(µ2-benzene-1,2-di-amine-κ2N:N')dicadmium], [Cd2(CH3COO)4(C6H8N2)2] n , (I), and poly[[(µ2-acetato-κ2O:O')(acetato-κ2O,O')(µ2-benzene-1,3-di-amine-κ2N:N')cadmium] hemihydrate], {[Cd(CH3COO)2(C6H8N2)]·0.5H2O} n , (II), have two-dimensional polymeric structures in which monomeric units are joined by bridging acetate and benzenedi-amine ligands. Each of the CdII ions has an O4N2 coordination environment. The coordination geometries of the symmetry-independent CdII ions are distorted octa-hedral and distorted trigonal anti-prismatic in (I) and distorted anti-prismatic in (II). Both compounds exhibit an intra-layer hydrogen-bonding network. In addition, the water of hydration in (II) is involved in inter-layer hydrogen bonding.

Poly[[tris-(µ2-acetato-κ(2) O:O')(4-chloro-benzene-1,2-di-amine-κN)(µ3-hydroxido)dizinc] ethanol monosolvate].

The title compound, {[Zn2(CH3CO2)3(OH)(C6H7ClN2)]·C2H5OH} n , has alternating octa-hedrally and tetra-hedrally coordinated Zn(2+) ions. The octa-hedral coordination sphere is composed of one N atom of the monodentate di-amino-chloro-benzene ligand, three acetate O atoms and two bridging hydroxide ligands. The tetra-hedral coordination sphere consists of three acetate O atoms and the hydroxide ligand. The zinc ions are bridged by acetate and hydroxide ligands. The result is a laddered-chain structure parallel to [100] with ethanol solvent mol-ecules occupying the space between the chains. The di-amine ligand chlorine substitutent is disordered over two equally populated positions as a result of a crystallographically imposed inversion center between adjacent ligands. The ethanol solvent mol-ecule exhibits disorder with the two components having refined occupancies of 0.696 (11) and 0.304 (11). O-H⋯O hydrogen bonds form between the hydroxide ligand and the ethanol solvent mol-ecule. N-H⋯O and N-H⋯N hydrogen bonding between the uncoordinated amine group and the acetate ligands and the coordinated amine group are also observed.

Hydrogen bonding in 4-nitrobenzene-1,2-diamine and two hydrohalide salts.

The structures of 4-nitrobenzene-1,2-diamine [C6H7N3O2, (I)], 2-amino-5-nitroanilinium chloride [C6H8N3O2(+)·Cl(-), (II)] and 2-amino-5-nitroanilinium bromide monohydrate [C6H8N3O2(+)·Br(-)·H2O, (III)] are reported and their hydrogen-bonded structures described. The amine group para to the nitro group in (I) adopts an approximately planar geometry, whereas the meta amine group is decidedly pyramidal. In the hydrogen halide salts (II) and (III), the amine group meta to the nitro group is protonated. Compound (I) displays a pleated-sheet hydrogen-bonded two-dimensional structure with R2(2)(14) and R4(4)(20) rings. The sheets are joined by additional hydrogen bonds, resulting in a three-dimensional extended structure. Hydrohalide salt (II) has two formula units in the asymmetric unit that are related by a pseudo-inversion center. The dominant hydrogen-bonding interactions involve the chloride ion and result in R4(2)(8) rings linked to form a ladder-chain structure. The chains are joined by N-H···Cl and N-H···O hydrogen bonds to form sheets parallel to (010). In hydrated hydrohalide salt (III), bromide ions are hydrogen bonded to amine and ammonium groups to form R4(2)(8) rings. The water behaves as a double donor/single acceptor and, along with the bromide anions, forms hydrogen bonds involving the nitro, amine, and ammonium groups. The result is sheets parallel to (001) composed of alternating R5(5)(15) and R6(4)(24) rings. Ammonium N-H···Br interactions join the sheets to form a three-dimensional extended structure. Energy-minimized structures obtained using DFT and MP2 calculations are consistent with the solid-state structures. Consistent with (II) and (III), calculations show that protonation of the amine group meta to the nitro group results in a structure that is about 1.5 kJ mol(-1) more stable than that obtained by protonation of the para-amine group. DFT calculations on single molecules and hydrogen-bonded pairs of molecules based on structural results obtained for (I) and for 3-nitrobenzene-1,2-diamine, (IV) [Betz & Gerber (2011). Acta Cryst. E67, o1359] were used to estimate the strength of the N-H···O(nitro) interactions for three observed motifs. The hydrogen-bonding interaction between the pairs of molecules examined was found to correspond to 20-30 kJ mol(-1).

Crystal structures of three lead(II) acetate-bridged di-amino-benzene coordination polymers.

Poly[tris-(acetato-κ(2) O,O')(µ2-acetato-κ(3) O,O':O)tetra-kis-(µ3-acetato-κ(4) O,O':O:O')bis-(benzene-1,2-di-amine-κN)tetra-lead(II)], [Pb4(CH3COO)8(C6H8N2)2] n , (I), poly[(acetato-κ(2) O,O')(µ3-acetato-κ(4) O,O':O:O')(4-chloro-benzene-1,2-diamine-κN)lead(II)], [Pb(CH3COO)2(C6H7ClN2)] n , (II), and poly[(κ(2) O,O')(µ3-acetato-κ(4) O,O':O:O')(3,4-di-amino-benzo-nitrile-κN)lead(II)], [Pb(CH3COO)2(C7H7N3)] n , (III), have polymeric structures in which monomeric units are joined by bridging acetate ligands. All of the Pb(II) ions exhibit hemidirected coordination. The repeating unit in (I) is composed of four Pb(II) ions having O6, O6N, O7 and O6N coordination spheres, respectively, where N represents a monodentate benzene-1,2-di-amine ligand and O acetate O atoms. Chains along [010] are joined by bridging acetate ligands to form planes parallel to (10-1). (II) and (III) are isotypic and have one Pb(II) ion in the asymmetric unit that has an O6N coordination sphere. Pb2O2 units result from a symmetry-imposed inversion center. Polymeric chains parallel to [100] exhibit hydrogen bonding between the amine and acetate ligands. In (III), additional hydrogen bonds between cyano groups and non-coordinating amines join the chains by forming R 2 (2)(14) rings.

An ortho-rhom-bic polymorph of 3,4-di-amino-benzo-nitrile.

The title compound, C7H7N3, is an ortho-rhom-bic polymorph that crystallizes in the space group Pca21. The previously reported monoclinic form [Geiger & Parsons (2013 ▶) Acta Cryst. E69, o452] crystallizes in the space group P21/c (Z = 4). In the crystal, two independent HN-H⋯N C hydrogen bonds link the mol-ecules into chains along the a-glide plane. Two further independent HN-H⋯NH2 hydrogen bonds join the chains, forming a three-dimensional network.

3,4-Diamino-benzonitrile.

The non-H atoms in the structure of the title mol-ecule, C7H7N3, are almost coplanar (r.m.s. deviation = 0.018 Å). The two amine groups each donate two and accept one weak N-H⋯N hydrogen bonds. N-H⋯N hydrogen bonding between the amine and nitrile groups results in chains parallel to [101] in the crystal structure. The chains are cross-linked by N-H⋯N hydrogen bonds between amine groups, giving rise to an infinite three-dimensional network.