Fragment optimization and elaboration strategies - the discovery of two lead series of PRMT5/MTA inhibitors from five fragment hits.

Here we describe the early stages of a fragment-based lead discovery (FBLD) project for a recently elucidated synthetic lethal target, the PRMT5/MTA complex, for the treatment of MTAP-deleted cancers. Starting with five fragment/PRMT5/MTA X-ray co-crystal structures, we employed a two-phase fragment elaboration process encompassing optimization of fragment hits and subsequent fragment growth to increase potency, assess synthetic tractability, and enable structure-based drug design. Two lead series were identified, one of which led to the discovery of the clinical candidate MRTX1719.

[Using the Treatment Passport to stimulate Advance Care Planning]. / Het gebruik van het Behandelpaspoort voor het stimuleren van Proactieve Zorgplanning.

Care recipients are becoming increasingly involved in the decision-making process for suitable treatments. Advance Care Planning (ACP) enables care recipients and healthcare professionals to timely think and discuss wishes and preferences for treatments, before a care recipient becomes limited by disease, cognitive problems or age. The Treatment Passport is developed by geriatric specialist Esther Bertholet and contains supportive questions to think about treatment wishes. This study aimed to evaluate the experiences of civilians and healthcare professionals with the usage of the Treatment Passport during ACP. Involved care organisations distributed the Treatment Passports to healthcare professionals, for example dementia casemanagers. Healthcare professionals then handed the Treatment passports to civilians, for example older persons. Thereafter, healthcare professionals (N=30) and civilians (N=80) filled out a questionnaire about the experiences with usage of the Treatment Passport. 65% of the civilians had a disease and a mean age 75.3 years. The Treatment Passport was considered a helpful tool to think and talk about treatment wishes. 57% of the healthcare professionals wants to keep using the Treatment Passport and 78% of the civilians would recommend it to others. Participants who found the passport of limited added value mostly had their own methods of discussing their treatment wishes.

Anti-tumor efficacy of a potent and selective non-covalent KRASG12D inhibitor.

Recent progress in targeting KRASG12C has provided both insight and inspiration for targeting alternative KRAS mutants. In this study, we evaluated the mechanism of action and anti-tumor efficacy of MRTX1133, a potent, selective and non-covalent KRASG12D inhibitor. MRTX1133 demonstrated a high-affinity interaction with GDP-loaded KRASG12D with KD and IC50 values of ~0.2 pM and <2 nM, respectively, and ~700-fold selectivity for binding to KRASG12D as compared to KRASWT. MRTX1133 also demonstrated potent inhibition of activated KRASG12D based on biochemical and co-crystal structural analyses. MRTX1133 inhibited ERK1/2 phosphorylation and cell viability in KRASG12D-mutant cell lines, with median IC50 values of ~5 nM, and demonstrated >1,000-fold selectivity compared to KRASWT cell lines. MRTX1133 exhibited dose-dependent inhibition of KRAS-mediated signal transduction and marked tumor regression (≥30%) in a subset of KRASG12D-mutant cell-line-derived and patient-derived xenograft models, including eight of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models. Pharmacological and CRISPR-based screens demonstrated that co-targeting KRASG12D with putative feedback or bypass pathways, including EGFR or PI3Kα, led to enhanced anti-tumor activity. Together, these data indicate the feasibility of selectively targeting KRAS mutants with non-covalent, high-affinity small molecules and illustrate the therapeutic susceptibility and broad dependence of KRASG12D mutation-positive tumors on mutant KRAS for tumor cell growth and survival.

Design and evaluation of achiral, non-atropisomeric 4-(aminomethyl)phthalazin-1(2H)-one derivatives as novel PRMT5/MTA inhibitors.

MRTX1719 is an inhibitor of the PRMT5/MTA complex and recently entered clinical trials for the treatment of MTAP-deleted cancers. MRTX1719 is a class 3 atropisomeric compound that requires a chiral synthesis or a chiral separation step in its preparation. Here, we report the SAR and medicinal chemistry design strategy, supported by structural insights from X-ray crystallography, to discover a class 1 atropisomeric compound from the same series that does not require a chiral synthesis or a chiral separation step in its preparation.

Design and Discovery of MRTX0902, a Potent, Selective, Brain-Penetrant, and Orally Bioavailable Inhibitor of the SOS1:KRAS Protein-Protein Interaction.

SOS1 is one of the major guanine nucleotide exchange factors that regulates the ability of KRAS to cycle through its "on" and "off" states. Disrupting the SOS1:KRASG12C protein-protein interaction (PPI) can increase the proportion of GDP-loaded KRASG12C, providing a strong mechanistic rationale for combining inhibitors of the SOS1:KRAS complex with inhibitors like MRTX849 that target GDP-loaded KRASG12C. In this report, we detail the design and discovery of MRTX0902─a potent, selective, brain-penetrant, and orally bioavailable SOS1 binder that disrupts the SOS1:KRASG12C PPI. Oral administration of MRTX0902 in combination with MRTX849 results in a significant increase in antitumor activity relative to that of either single agent, including tumor regressions in a subset of animals in the MIA PaCa-2 tumor mouse xenograft model.

Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers.

The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP-deleted cancers. Here, we report the discovery of development candidate MRTX1719. MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP-deleted cells compared to MTAP-wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP-deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)phthalazin-1(2H)-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719.

Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor.

KRASG12D, the most common oncogenic KRAS mutation, is a promising target for the treatment of solid tumors. However, when compared to KRASG12C, selective inhibition of KRASG12D presents a significant challenge due to the requirement of inhibitors to bind KRASG12D with high enough affinity to obviate the need for covalent interactions with the mutant KRAS protein. Here, we report the discovery and characterization of the first noncovalent, potent, and selective KRASG12D inhibitor, MRTX1133, which was discovered through an extensive structure-based activity improvement and shown to be efficacious in a KRASG12D mutant xenograft mouse tumor model.

Evaluation of ß-Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure.

ß-Amino acids have a backbone that is expanded by one carbon atom relative to α-amino acids, and ß residues have been investigated as subunits in protein-like molecules that adopt discrete and predictable conformations. Two classes of ß residue have been widely explored in the context of generating α-helix-like conformations: ß3 -amino acids, which are homologous to α-amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five-membered ring, such the one derived from trans-2-aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their ß3  homologues within an α-helix-forming sequence generally causes a decrease in conformational stability. Use of a ring-constrained ß residue, however, can offset the destabilizing effect of αâ†’ß substitution. Here we extend the study of αâ†’ß substitutions, involving both ß3 and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two-, three-, or four-residue ß-hairpin loop, and we evaluate αâ†’ß replacements at each loop position for each variant. By referral to the ϕ,ψ angles of the native structure, one can choose a stereochemically appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three ß-containing Pin1 WW domain variants show that a native-like tertiary structure is maintained in each case.

Toward a Soluble Model System for the Amyloid State.

The formation and deposition of amyloids is associated with many diseases. ß-Sheet secondary structure is a common feature of amyloids, but the packing of sheets against one another is distinctive relative to soluble proteins. Standard methods that rely on perturbing a polypeptide's sequence and evaluating impact on folding can be problematic for amyloid aggregates because a single sequence can adopt multiple conformations and diverse packing arrangements. We describe initial steps toward a minimum-sized, soluble model system for the amyloid state that supports comparisons among sequence variants. Critical to this goal is development of a new linking strategy to enable intersheet association mediated by side chain interactions, which is characteristic of the amyloid state. The linker design we identified should ultimately support exploration of relationships between sequence and amyloid state stability for specific strand-association modes.

Hypereosinophilia in Children and Adults: A Retrospective Comparison.

BACKGROUND: The differential diagnosis of hypereosinophilia is broad and includes asthma, atopic disease, drug hypersensitivity, parasitic infection, connective tissue disorders, malignancy, and rare hypereosinophilic disorders. Hypereosinophilia in children has not been well characterized to date. OBJECTIVE: The objective of this study was to identify the common causes of marked eosinophilia in children and to characterize and compare the clinical symptoms at presentation, laboratory findings, final diagnosis, and therapeutic responses between children and adults with hypereosinophilic syndromes. METHODS: A retrospective analysis of consecutive subjects evaluated for unexplained eosinophilia ≥ 1.5 × 10(9)/L was conducted. All subjects underwent standardized clinical and laboratory evaluations with yearly follow-up. Clinical and laboratory parameters, final diagnoses, treatment responses, and outcomes were assessed. Medians and proportions were compared using Mann-Whitney U and Fisher Exact tests, respectively. RESULTS: Of the 291 subjects evaluated, 37 (13%) were children and 254 were adults (87%). Whereas the frequencies of clinical hypereosinophilic syndrome (HES) variants were similar between children and adults, primary immunodeficiency was a more common secondary cause of HES in children (5% vs 0.4% in adults). Excluding subjects with treatable secondary causes, the median peak absolute eosinophil count was increased in pediatric subjects (9376 vs 5543/µL; P = .002), and children had more gastrointestinal complaints (62% vs 34%; P = .003) and less pulmonary involvement (34% vs 59%; P = .01) than adults. Despite these differences, corticosteroid responsiveness and overall prognosis were similar between the 2 groups. CONCLUSIONS: Although children with HES often present with higher peak eosinophil counts than adults, the differential diagnosis, clinical characteristics, and prognosis of HES are similar in the 2 groups.

Quasiracemate Crystal Structures of Magainin 2 Derivatives Support the Functional Significance of the Phenylalanine Zipper Motif.

Quasiracemic crystallography has been used to explore the significance of homochiral and heterochiral associations in a set of host-defense peptide derivatives. The previously reported racemic crystal structure of a magainin 2 derivative displayed a homochiral antiparallel dimer association featuring a "phenylalanine zipper" notable for the dual roles of phenylalanines in mediating dimerization and formation of an exposed hydrophobic swath. This motif is seen as well in two new quasiracemate crystals that contain the d form of the magainin 2 derivative along with an l-peptide in which one Ala has been replaced by a ß-amino acid residue. This structural trend supports the hypothesis that the Phe zipper motif has functional significance.

Targeting diverse protein-protein interaction interfaces with α/ß-peptides derived from the Z-domain scaffold.

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and ß-amino acid residues ("α/ß-peptides") that mimic several peptides derived from the three-helix bundle "Z-domain" scaffold. We show that α/ß-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/ß-peptide inhibits the VEGF165-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain-mimetic α/ß-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/ß-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

Marked and persistent eosinophilia in the absence of clinical manifestations.

BACKGROUND: Although most patients with hypereosinophilic syndromes (HES) present with clinical signs and symptoms attributable to eosinophilic tissue infiltration, some untreated patients remain asymptomatic or have signs and symptoms, such as allergic rhinitis, for which the relationship to peripheral eosinophilia is unclear (hypereosinophilia of unknown significance [HEUS]). OBJECTIVE: To identify and characterize subjects with HEUS of 5 years duration or more as compared to untreated patients with symptomatic HES and healthy normal volunteers. METHODS: All subjects with eosinophilia underwent yearly evaluation, including a standardized clinical evaluation, whole blood flow cytometry to assess lymphocyte subsets and eosinophil activation, and serum collection. Peripheral blood mononuclear cells were cultured overnight with and without phorbol 12-myristate 13-acetate/ionomycin. Cytokines and chemokines were measured in serum and cell supernatants, and mRNA expression was assessed by using quantitative real-time PCR. RESULTS: Eight of the 210 subjects referred for the evaluation of eosinophilia (absolute eosinophil count [AEC] > 1500/µL) met the criteria for HEUS of 5 years duration or more (range, 7-29 years). Peak eosinophil count and surface expression of eosinophil activation markers were similar in subjects with HEUS and in untreated subjects with platelet-derived growth factor alpha-negative HES (n = 28). Aberrant or clonal T-cell populations were identified in 50% of the subjects with HEUS as compared to 29% of the subjects with HES (P = .12). Increased levels of IL-5, GM-CSF, IL-9, and IL-17A were also comparable in subjects with HEUS and HES. Serum levels of IgE and IL-13 were significantly increased only in subjects with HES. CONCLUSIONS: A small number of patients with persistent peripheral eosinophilia (AEC > 1500/µL) appear to have clinically benign disease.