Components of the DNA mismatch repair (MMR) pathway modulate somatic CAG-repeat expansion at the huntingtin (HTT) locus and alter the age of clinical motor diagnosis and progression of Huntington's disease (HD). MSH3, which encodes a subunit of MutSβ (MSH2-MSH3), has been identified as a genetic modifier of age at onset of multiple clinical phenotypes in people with HD (PwHD). MutSβ interacts with MutL endonucleases (MutLα, MLH1-PMS2 and MutLγ MLH1-MLH3) in a ternary complex with DNA to initiate MMR. Disruption of the MLH1-MSH3 protein-protein interaction (PPI) represents a potential therapeutic strategy to inhibit MMR and limit somatic CAG-repeat expansion. Leveraging the MLH1 Interaction Protein (MIP) Box sequence and the structural information of the hMLH1 C-terminal domain (CTD) bound to an MSH3-derived peptide (21 QAVLSRFFQ 29), we designed and optimized potent linear and cyclic peptides targeting the MLH1-MSH3 interface. Dimerization of the optimized cyclic peptide (compound 52) enhanced binding affinity, achieving double-digit nanomolar potency in a MutSβ:MutLα:DNA (MLH1-MSH3-DNA) ternary complex assay in the presence of 100 μM of ATP (compound 52 IC50 = 87 nM, compound 56 IC50 = 32 nM). Evaluation in an in vitro MMR DNA repair assay using cellular nuclear extracts from MMR-proficient and-deficient cell lines demonstrated that our lead compound (56) effectively inhibits MutSβ/MutLα-dependent DNA repair in human cells. These results highlight cyclic peptide-mediated disruption of MLH1-MSH3 as a promising approach to modulate MMR activity and possibly mitigate somatic CAG-repeat expansion in HD.

Orthosteric inhibitors of the human heterodimeric DNA mismatch repair complex MutSβ were identified by high-throughput screening. Following extensive hit confirmation to remove false positives, two series were found to give consistent activity free of likely artefactual effects. Extensive hit profiling confirmed an ATP-competitive mode of action and resulted in our obtaining the first reported X-ray and cryo-EM structures of small molecule inhibitors of MutSβ occupying the ATP-binding site of MSH3.

Peptide 1, a C18 fatty acid-modified single-chain relaxin analogue, was recently identified as a potent, selective, and long-lasting relaxin family peptide receptor 1 (RXFP1) agonist. Further advanced pharmacokinetic profiling of this compound highlighted elevated levels of oxidative metabolism occurring in dogs and mini pigs but only marginally in rats. This study aimed to design long-lasting relaxin analogues with increased stability against metabolic oxidation while securing subnanomolar RXFP1 potency. Key structural elements, including fatty acid chain length, attachment position, and linker structure, were modified to reduce oxidative metabolism and improve pharmacokinetic parameters. Additionally, incorporating α-methyl lysine (Mly) at position 30, alongside other selective sequence mutations, resulted in several analogues with subnanomolar RXFP1 potency and improved duration of action compared to 1. Compound 21 (R2R01) was then selected as a candidate for an in-depth characterization. It is currently undergoing phase 2 clinical development for renal and cardiovascular diseases.

FAN1 is an endo- and exo-nuclease involved in DNA and interstrand crosslink repair. Genome-wide association studies of people with Huntington's disease revealed a strong association between the FAN1 R507H mutation and early disease onset, however the underlying mechanism(s) remains unclear. FAN1 has previously been implicated in modulating triplet repeat expansion in a PCNA dependent manner. To examine the role of PCNA on FAN1 activation, we solved the cryo-EM structures of a PCNA-FAN1-DNA complex. Our findings reveal that the FAN1 R507 residue directly interacts with PCNA D232. Biophysical interaction studies demonstrated that FAN1 enhances the binding affinity of PCNA for DNA, a synergistic effect disrupted in mutants carrying the R507H mutation. In contrast, PCNA does not affect the affinity of FAN1 for DNA but does modulate FAN1 activity upon ternary complex formation. The weakened and functionally altered FAN1 R507H-PCNA-DNA complex may partly impair the FAN1-mediated repair of CAG extrahelical extrusions, providing a potential explanation for the mutation's role in accelerating disease progression.

Human and mouse genetic studies have demonstrated a role for DNA mismatch repair (MMR) molecular machines in modulating the rate of somatic expansion of the huntingtin (HTT) CAG repeats, and onset and progression of Huntington's Disease (HD). MutS beta, a key component of the MMR pathway, is a heterodimeric protein of MSH2 and MSH3 that recognizes and initiates the repair of extrahelical DNA extrusions. Loss-of-function of mouse Msh3 and reduced-expression alleles of human MSH3 lead to slower rates of somatic expansion and delayed disease onset in humans, signifying MSH3 as a promising therapeutic target for HD. Here we report biochemical and cryo-electron microscopy analyses of human MutS beta, demonstrating MutS beta undergoes conformational changes induced by nucleotide and DNA binding. We present multiple conformations of MutS beta including the DNA-free MutS beta compatible with precisely complementary base-paired homoduplex DNA binding, two distinct structures of MutS beta bound to (CAG)2 DNA, a sliding clamp form and a DNA-unbound, ATP-bound conformation. Along with evidence for novel conformational states adopted by MutS beta to initiate the MMR cascade, these structures provide a foundation for structure-guided drug discovery.

Huntington's disease is a neurodegenerative disorder associated with a polyglutamine expansion within the first exon of the huntingtin protein (HTT exon 1). This mutation results in HTT dysfunction and the production of N-terminal HTT aggregates. The dimerization of the HTT exon 1 fragment through self-association of the first 17 residues (N17) is considered the initial step in the HTT exon 1 aggregation pathway. The association of N17 with membranes has been proposed to catalyze aggregation by increasing the local concentration of exon 1, and post-translational modifications (PTMs) in N17 are known to influence membrane interaction and the aggregation rate of exon 1. To elucidate the influence of N17 PTMs on both self-association and membrane interaction, thereby gaining insight into HTT function and exon 1 aggregation, we used solution nuclear magnetic resonance and circular dichroism spectroscopies to address loss of initial methionine, subsequent acetylation, and phosphorylation of threonine and serines. Our findings indicate that modifications to N17 that enhance helicity correspond to increased self-association and membrane interaction. We then conducted X-ray crystallographic studies that led to a proposed HTT exon 1 dimerization model consistent with the association of N17 dimers. This provides insight into the impact of PTMs on HTT aggregation. The experimental methods and N17 self-association model we describe may serve as a foundation for further experiments exploring the influence of N17 PTMs on HTT function and pathogenicity.

Huntington’s disease (HD) is fatal neurodegenerative disorder caused by the expansion of a CAG-repeat tract in the huntingtin ( HTT ) gene. Human and mouse genetics studies have demonstrated a role for DNA mismatch repair (MMR) proteins which control the rate of somatic expansion of the HTT CAG repeat and disease onset and progression. MutSβ, a key member of the MMR pathway, is a heterodimeric protein of MSH2 and MSH3 that recognizes and initiates the repair of small insertion or deletion DNA loop outs. Both mouse Msh3 loss-of-function and reduced-expression alleles of human MSH3 lead to slower rates of somatic expansion in the HTT CAG tract and a delay of disease onset and progression, signifying MSH3 as a promising drug target for HD. Structural biology studies of MutSβ are informative for mechanism, protein structure-function relationships, and guiding small-molecule drug design. Here we report biochemical and cryo-electron microscopy analyses of human MutSβ ensembles, revealing that MutSβ undergoes multiple conformational changes in response to binding and release of nucleotides and DNA. The DNA-free MutSβ-ADP complex adopts an open conformation that is compatible with DNA binding. The conformation of MutSβ in the (CAG) 2 DNA-bound open structure most closely resembles the recently identified low-affinity state of MutSα, compared to the canonical mismatch-bound conformation. The homoduplex-bound and DNA-unbound MutSβ-ATP structures show that MutSβ undergoes an ATP-dependent conformational change towards sliding clamp forms. This study provides a comprehensive understanding of the structural conformational dynamics of MutSβ, insights into the MMR cascade, and a foundation for structure-guided drug discovery.

Orthosteric inhibitors of the human heterodimeric DNA mismatch repair complex MutSbeta were identified by high-throughput screening. Following extensive hit confirmation to remove false positives, two series were found to give consistent activity free of likely artefactual effects. Extensive hit profiling confirmed an ATP-competitive mode of action, and X-ray crystallography showed the inhibitors occupying the ATP-binding site of MSH3.

We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.

Huntington’s disease (HD) is caused by the expansion of a polyglutamine (polyQ)-encoding tract in exon 1 of the huntingtin gene to greater than 35 CAG repeats. It typically has a disease course lasting 15–20 years, and there are currently no disease-modifying therapies available. Thus, there is a need for faithful mouse models of HD to use in preclinical studies of disease mechanisms, target validation, and therapeutic compound testing. A large variety of mouse models of HD were generated, none of which fully recapitulate human disease, complicating the selection of appropriate models for preclinical studies. Here, we present the urinary liquid chromatography–high-resolution mass spectrometry analysis employed to identify metabolic alterations in transgenic R6/2 and zQ175DN knock-in mice. In R6/2 mice, the perturbation of the corticosterone metabolism and the accumulation of pyrraline, indicative of the development of insulin resistance and the impairment of pheromone excretion, were observed. Differently from R6/2, zQ175DN mice showed the accumulation of oxidative stress metabolites. Both genotypes showed alterations in the tryptophan metabolism. This approach aims to improve our understanding of the molecular mechanisms involved in HD neuropathology, facilitating the selection of appropriate mouse models for preclinical studies. It also aims to identify potential biomarkers specific to HD.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. We report the design of a series of HTT pre-mRNA splicing modulators that lower huntingtin (HTT) protein, including the toxic mutant huntingtin (mHTT), by promoting insertion of a pseudoexon containing a premature termination codon at the exon 49-50 junction. The resulting transcript undergoes nonsense-mediated decay, leading to a reduction of HTT mRNA transcripts and protein levels. The starting benzamide core was modified to pyrazine amide and further optimized to give a potent, CNS-penetrant, and orally bioavailable HTT-splicing modulator 27. This compound reduced canonical splicing of the HTT RNA exon 49-50 and demonstrated significant HTT-lowering in both human HD stem cells and mouse BACHD models. Compound 27 is a structurally diverse HTT-splicing modulator that may help understand the mechanism of adverse effects such as peripheral neuropathy associated with branaplam.

Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3-778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.

Lipidation, a common strategy to improve half-life of therapeutic peptides, affects their tendency to oligomerize, their interaction with plasmatic proteins, and their catabolism. In this work, we have leveraged the use of NMR and SPR spectroscopy to elucidate oligomerization propensity and albumin interaction of different analogs of the two marketed lipidated GLP-1 agonists liraglutide and semaglutide. As most lipidated therapeutic peptides are administered by subcutaneous injection, we have also assessed in vitro their catabolism in the SC tissue using the LC-HRMS-based SCiMetPep assay. We observed that oligomerization had a shielding effect against catabolism. At the same time, binding to albumin may provide only limited protection from proteolysis due to the higher unbound peptide fraction present in the subcutaneous compartment with respect to the plasma. Finally, identification of catabolites in rat plasma after SC dosing of semaglutide showed a good correlation with the in vitro data, with Tyr19-Leu20 being the major cleavage site. Early characterization of the complex interplay between oligomerization, albumin binding, and catabolism at the injection site is essential for the synthesis of lipidated peptides with good pharmacokinetic profiles.

A new series of in vitro potent and highly selective histone methyl transferase enzyme G9a inhibitors was obtained. In particular, compound 2a, one the most potent G9a inhibitor identified, was endowed with >130-fold selectivity over GLP and excellent ligand efficiency. Therefore, it may represent a valuable tool compound to validate the role of highly selective G9a inhibitors in different pathological conditions. When 2a was characterized in vitro in cellular models of skeletal muscle differentiation, a relevant increase of myofibers' size and reduction of the fibroadipogenic infiltration were observed, further confirming the therapeutic potential of selective G9a inhibitors for the treatment of Duchenne muscle dystrophy.

Despite beneficial effects in acute heart failure, the full therapeutic potential of recombinant relaxin-2 has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. A multiparametric optimization of the relaxin B-chain led to the identification of single chain lipidated peptide agonists of RXFP1 like SA10SC-RLX with subcutaneous bioavailability and extended half-life. SA10SC-RLX has sub nanomolar activity on cells expressing human RXFP1 and molecular modeling associated with the study of different RXFP1 mutants was used to decipher the mechanism of SA10SC-RLX interaction with RXFP1. Telemetry was performed in rat where SA10SC-RLX was able to engage RXFP1 after subcutaneous administration without tachyphylaxis after repeated dosing. Renal blood flow was then used as a translational model to evaluate RXFP1 activation. SA10SC-RLX increased renal blood flow and decreased renal vascular resistance in rats as reported for relaxin in humans. In conclusion, SA10SC-RLX mimics relaxin activity in in vitro and in vivo models of acute RXFP1 engagement. SA10SC-RLX represents a new class of long-lasting RXFP1 agonist, suitable for once daily subcutaneous administration in patients and potentially paving the way to new treatments for chronic fibrotic and cardiovascular diseases.