Identification and Optimization of RNA-Splicing Modulators as Huntingtin Protein-Lowering Agents for the Treatment of Huntington's Disease.

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.

Developing HDAC4-Selective Protein Degraders To Investigate the Role of HDAC4 in Huntington's Disease Pathology.

Huntington's disease (HD) is a lethal autosomal dominant neurodegenerative disorder resulting from a CAG repeat expansion in the huntingtin (HTT) gene. The product of translation of this gene is a highly aggregation-prone protein containing a polyglutamine tract >35 repeats (mHTT) that has been shown to colocalize with histone deacetylase 4 (HDAC4) in cytoplasmic inclusions in HD mouse models. Genetic reduction of HDAC4 in an HD mouse model resulted in delayed aggregation of mHTT, along with amelioration of neurological phenotypes and extended lifespan. To further investigate the role of HDAC4 in cellular models of HD, we have developed bifunctional degraders of the protein and report the first potent and selective degraders of HDAC4 that show an effect in multiple cell lines, including HD mouse model-derived cortical neurons. These degraders act via the ubiquitin-proteasomal pathway and selectively degrade HDAC4 over other class IIa HDAC isoforms (HDAC5, HDAC7, and HDAC9).

Structure-Based Exploration of Selectivity for ATM Inhibitors in Huntington's Disease.

Our group has recently shown that brain-penetrant ataxia telangiectasia-mutated (ATM) kinase inhibitors may have potential as novel therapeutics for the treatment of Huntington's disease (HD). However, the previously described pyranone-thioxanthenes (e.g., 4) failed to afford selectivity over a vacuolar protein sorting 34 (Vps34) kinase, an important kinase involved with autophagy. Given that impaired autophagy has been proposed as a pathogenic mechanism of neurodegenerative diseases such as HD, achieving selectivity over Vps34 became an important objective for our program. Here, we report the successful selectivity optimization of ATM over Vps34 by using X-ray crystal structures of a Vps34-ATM protein chimera where the Vps34 ATP-binding site was mutated to approximate that of an ATM kinase. The morpholino-pyridone and morpholino-pyrimidinone series that resulted as a consequence of this selectivity optimization process have high ATM potency and good oral bioavailability and have lower molecular weight, reduced lipophilicity, higher aqueous solubility, and greater synthetic tractability compared to the pyranone-thioxanthenes.

Evaluation of 5-(Trifluoromethyl)-1,2,4-oxadiazole-Based Class IIa HDAC Inhibitors for Huntington's Disease.

Using an iterative structure-activity relationship driven approach, we identified a CNS-penetrant 5-(trifluoromethyl)-1,2,4-oxadiazole (TFMO, 12) with a pharmacokinetic profile suitable for probing class IIa histone deacetylase (HDAC) inhibition in vivo. Given the lack of understanding of endogenous class IIa HDAC substrates, we developed a surrogate readout to measure compound effects in vivo, by exploiting the >100-fold selectivity compound 12 exhibits over class I/IIb HDACs. We achieved adequate brain exposure with compound 12 in mice to estimate a class I/IIb deacetylation EC50, using class I substrate H4K12 acetylation and global acetylation levels as a pharmacodynamic readout. We observed excellent correlation between the compound 12 in vivo pharmacodynamic response and in vitro class I/IIb cellular activity. Applying the same relationship to class IIa HDAC inhibition, we estimated the compound 12 dose required to inhibit class IIa HDAC activity, for use in preclinical models of Huntington's disease.

Optimization of Potent and Selective Ataxia Telangiectasia-Mutated Inhibitors Suitable for a Proof-of-Concept Study in Huntington's Disease Models.

Genetic and pharmacological evidence indicates that the reduction of ataxia telangiectasia-mutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington's disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here, we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein liability while maintaining potency and selectivity. Here, we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in the mouse brain and an understandable pharmacokinetic/PD (PK/PD) relationship. Compound 17 engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in the mouse brain. Furthermore, compound 17 protects against mHTT (Q73)-induced cytotoxicity in a cortical-striatal cell model of HD.

Development and characterization of a CNS-penetrant benzhydryl hydroxamic acid class IIa histone deacetylase inhibitor.

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ∼150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC50 levels for ∼8 h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.

Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors.

Potent and selective class IIa HDAC tetrasubstituted cyclopropane hydroxamic acid inhibitors were identified with high oral bioavailability that exhibited good brain and muscle exposure. Compound 14 displayed suitable properties for assessment of the impact of class IIa HDAC catalytic site inhibition in preclinical disease models.

Inter- and intramolecular annulation strategies to a cyclopentanone building block containing an all-carbon quaternary stereogenic center.

Synthesis of (S)-2-methyl-3-fluorophenyl cyclopentanone methyl ester (1S)-1 has been achieved by both inter- and intramolecular alkylation reactions on multigram scale, using chiral pool reagents. The intramolecular variant is a novel example of a chiral bis-electrophile reacting with a carbon nucleophile to form an enantiomerically pure all-carbon quaternary center.

Design, synthesis, and biological evaluation of potent and selective class IIa histone deacetylase (HDAC) inhibitors as a potential therapy for Huntington's disease.

Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntington's disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a "closed-loop" form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.

In silico scaffold evaluation and solid phase approach to identify new gelatinase inhibitors.

Among matrix metalloproteinases (MMPs), gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) play a key role in a number of physiological processes such as tissue repair and fibrosis. Many evidences point out their involvement in a series of pathological events, such as arthritis, multiple sclerosis, cardiovascular diseases, inflammatory processes and tumor progression by degradation of the extracellular matrix. To date, the identification of non-specific MMP inhibitors has made difficult the selective targeting of gelatinases. In this work we report the identification, design and synthesis of new gelatinase inhibitors with appropriate drug-like properties and good profile in terms of affinity and selectivity. By a detailed in silico protocol and innovative and versatile solid phase approaches, a series of 4-thiazolydinyl-N-hydroxycarboxyamide derivatives were identified. In particular, compounds 9a and 10a showed a potent inhibitory activity against gelatinase B and good selectivity over the other MMP considered in this study. The identified compounds could represent novel potential candidates as therapeutic agents.

Thiopyrophosphoantigens: solid-phase synthesis and in vitro characterization of a new class of Vgamma9 Vdelta2 T cells activators.

The Vgamma9 Vdelta2 T cells mediate rapid, innate-like immune responses to pathogens and are important in several key immunoregulatory pathways, including those involved in infections and tumor development. Vgamma9 Vdelta2 T cells respond to low molecular weight isoprenoid phosphoantigens; the prototypic stimulatory compound is isopentenylpyrophosphate (IPP), an alkylphosphate intermediate of mevalonate metabolism that elicits proliferative, cytotoxic, and cytokine secretion responses. We studied the replacement of the pyrophosphate moiety with the thiopyrophosphate bioisostere, synthesizing thioanalogues of IPP and 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP, the most potent natural antigen known to date). Once their in vitro efficacy and stability had been demonstrated, we synthesized a small library of compounds through the development of an innovative solid-phase strategy. Biological results confirmed thioHMBPP to be the best compound of this first series. Future aims are (i) the exploitation of the parallel solid-phase strategy to further explore structure-activity relationships of this new class of synthetic antigens and (ii) the determination of the PK/PD profile of thioHMBPP.

Dimerization inhibitors of HIV-1 protease based on a bicyclic guanidinium subunit.

Original inhibitors of HIV-1 protease based on a chiral bicyclic guanidinium scaffold linked to short peptidic mimics of the terminal protease sequences and to a lipophilic group were designed. These inhibitors prevent dimerization of the native protease by an interfacial structure at the highly conserved antiparallel beta-strand involving both the N and C termini that substantially account for dimerization. The preorganized guanidinium spacer introduces additional electrostatic hydrogen-bonding interactions with the C-terminal Phe-99 carboxylate. Lipophilic residues linked to side chains and the guanidinium scaffold are essential for dimerization inhibition as ascertained by Zhang kinetics (4, K(id) = 290 nM; 6 or 6', K(id) = 150 nM; 8, K(id) = 400 nM) combined with a circular dichroism study on the enzyme thermal stability. Remarkably, less hydrophobic compounds result in mixed dimerization (1a and 3) or active site inhibitors (5). Removal of the guanidinium hydrophobic groups leads to less active or inactive ligands.

Guanidinium receptors as enantioselective amino acid membrane carriers.

A number of artificial carriers for the transport of zwitterionic aromatic amino acids across bulk model membranes (U-tube type) have been prepared and evaluated. 1,2-Dichloroethane and dichloromethane were employed in the organic phase. All compounds are based on a bicyclic chiral guanidinium scaffold that ideally complements the carboxylate function. The guanidinium central moiety was attached to crown ethers or lasalocid A as specific subunits for ammonium recognition as well as to aromatic or hydrophobic residues to evaluate their potential interaction with the side chains of the guest amino acids. The subunits were linked to the guanidinium through ester or amide connectors. Amides were found to be better carriers than esters, though less enantioselective. On the other hand, crown ethers were superior to lasalocid derivatives. As expected, transport rates were dependent on the carrier concentration in the liquid membrane. Reciprocally, enantioselectivities were much higher at lower carrier concentrations. The results show that our previously proposed three-point binding model (J. Am. Chem. Soc. 1992, 114, 1511-1512), involving the participation of the aromatic or hydrophobic residue to interact with the side chains of the amino acid guest, is unnecessary to explain the high enantioselectivities observed. Molecular dynamics fully support a two-point model involving only the guanidinium and crown ether moieties. These molecules constitute the first examples of chiral selectors for underivatized amino acids acting as carriers under neutral conditions.

Enantioselective transport by a steroidal guanidinium receptor.

The cationic steroidal receptors 9 and 11 have been synthesized from cholic acid 3. Receptor 9 extracts N-acetyl-alpha-amino acids from aqueous media into chloroform with enantioselectivities (L:D) of 7-10:1. The lipophilic variant 11 has been employed for the enantioselective transport of N-acetylphenylalanine, a) through dichloromethane (DCM) and dichloroethane (DCE) bulk liquid membranes (U-tube apparatus), and b) through 2.5% (v/v) octanol/hexane via hollow fibre membrane contactors. Significant enantioselectivities and multiple turnovers were observed for both types of apparatus.