Small Molecule Therapeutics Research Articles

Biocatalysis in Drug Design: Engineered Reductive Aminases (RedAms) Are Used to Access Chiral Building Blocks with Multiple Stereocenters.

Novel building blocks are in constant demand during the search for innovative bioactive small molecule therapeutics by enabling the construction of structure-activity-property-toxicology relationships. Complex chiral molecules containing multiple stereocenters are an important component in compound library expansion but can be difficult to access by traditional organic synthesis. Herein, we report a biocatalytic process to access a specific diastereomer of a chiral amine building block used in drug discovery. A reductive aminase (RedAm) was engineered following a structure-guided mutagenesis strategy to produce the desired isomer. The engineered RedAm (IR-09 W204R) was able to generate the (S,S,S)-isomer 3 in 45% conversion and 95% ee from the racemic ketone 2. Subsequent palladium-catalyzed deallylation of 3 yielded the target primary amine 4 in a 73% yield. This engineered biocatalyst was used at preparative scale and represents a potential starting point for further engineering and process development.

Multi-structural molecular docking (MOD) combined with molecular dynamics reveal the structural requirements of designing broad-spectrum inhibitors of SARS-CoV-2 entry to host cells

New variants of SARS-CoV-2 that can escape immune response continue to emerge. Consequently, there is an urgent demand to design small molecule therapeutics inhibiting viral entry to host cells to reduce infectivity rate. Despite numerous in silico and in situ studies, the structural requirement of designing viral-entry inhibitors effective against multiple variants of SARS-CoV-2 has yet to be described. Here we systematically screened the binding of various natural products (NPs) to six different SARS-CoV-2 receptor-binding domain (RBD) structures. We demonstrate that Multi-structural Molecular Docking (MOD) combined with molecular dynamics calculations allowed us to predict a vulnerable site of RBD and the structural requirement of ligands binding to this vulnerable site. We expect that our findings lay the foundation for in silico screening and identification of lead molecules to guide drug discovery into designing new broad-spectrum lead molecules to counter the threat of future variants of SARS-CoV-2.

Distinct chemical environments in biomolecular condensates.

Diverse mechanisms have been described for selective enrichment of biomolecules in membrane-bound organelles, but less is known about mechanisms by which molecules are selectively incorporated into biomolecular assemblies such as condensates that lack surrounding membranes. The chemical environments within condensates may differ from those outside these bodies, and if these differed among various types of condensate, then the different solvation environments would provide a mechanism for selective distribution among these intracellular bodies. Here we use small molecule probes to show that different condensates have distinct chemical solvating properties and that selective partitioning of probes in condensates can be predicted with deep learning approaches. Our results demonstrate that different condensates harbor distinct chemical environments that influence the distribution of molecules, show that clues to condensate chemical grammar can be ascertained by machine learning and suggest approaches to facilitate development of small molecule therapeutics with optimal subcellular distribution and therapeutic benefit.

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

DEVELOPMENT OF GLIEXP; A HIGH-THROUGHPUT EX VIVO DRUG SCREENING PLATFORM FOR GLIOBLASTOMA

Abstract AIMS With diminishing returns and common clinical failure rates from traditional preclinical and animal-based drug discovery strategies, emphasis is being placed on alternative drug discovery platforms, such as ex vivo approaches. Such approaches represent a departure from both preclinical animal-based models and more traditional clinical-based strategies and aim to address intra/inter-patient variability at an earlier stage of drug discovery. Additionally, they could also offer precise treatment stratification for patients within a week of surgery to direct a tailored treatment course. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy- resistant glioma stem cell (GSC) niches, and where historic preclinical models have failed to generate new therapies leading to 50-year stagnant dire survival rates of around 15 months post-diagnosis. METHOD We have developed and optimized a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. RESULTS As a proof-of-concept for GliExP, we have optimized standard-of-care chemoradiotherapy responses which can correctly predict MGMT status based on Temozolomide sensitivity. We have screened 30+ small molecule therapeutics and preclinical compounds against 18 different patient’s tumours, including multi-region spatial het- erogeneity sampling from several individual tumours. Drug responses are further correlated to transcriptomic and genetic profiles. CONCLUSIONS Our data provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with standard-of-care therapies as an important preclinical model to triage experimental therapeutics for clinical translation, delivering rapid identification of effective treatment strategies for individual gliomas.

MicroRNA-194-3p impacts autophagy and represses rotavirus replication via targeting silent information regulator 1

BackgroundRotavirus (RV) is the main cause of serious diarrhea in infants and young children worldwide. Numerous studies have demonstrated that RV use host cell mechanisms to motivate their own stabilization and multiplication by degrading, enhancing, or hijacking microRNAs (miRNAs). Therefore, exploring the molecular mechanisms by which miRNAs motivate or restrain RV replication by controlling different biological processes, including autophagy, will help to better understand the pathogenesis of RV development. This study mainly explored the effect of miR-194-3p on autophagy after RV infection and its underlying mechanism of the regulation of RV replication.MethodsCaco-2 cells were infected with RV and used to measure the expression levels of miR-194-3p and silent information regulator 1 (SIRT1). After transfection with plasmids and RV infection, viral structural proteins, RV titer, cell viability, and autophagy-linked proteins were tested. The degree of acetylation of p53 was further investigated. A RV-infected neonatal mouse model was constructed in vivo and was evaluated for diarrhea symptoms and lipid droplet formation.ResultsThe results showed that miR-194-3p was reduced but SIRT1 was elevated after RV infection. Elevation of miR-194-3p or repression of SIRT1 inhibited RV replication through the regulation of autophagy. The overexpression of SIRT1 reversed the effects of miR-194-3p on RV replication. The upregulation of miR-194-3p or the downregulation of SIRT1 repressed RV replication in vivo. MiR-194-3p targeted SIRT1 to decrease p53 acetylation.ConclusionThese results were used to determine the mechanism of miR-194-3p in RV replication, and identified a novel therapeutic small RNA molecule that can be used against RV.

Enzyme-Responsive Nanoparticles for the Targeted Delivery of an MMP Inhibitor to Acute Myocardial Infarction.

Herein, we have developed a drug-loaded matrix metalloproteinase (MMP)-responsive micellar nanoparticle (NP) intended for minimally invasive intravenous injection during the acute phase of myocardial infarction (MI) and prolonged retention in the heart for small-molecule drug delivery. Peptide-polymer amphiphiles (PPAs) bearing a small-molecule MMP inhibitor (MMPi), PD166793, were synthesized via ring-opening metathesis polymerization (ROMP) and formulated into spherical micelles by transitioning to aqueous solution. The resulting micellar NPs underwent MMP-induced aggregation, demonstrating enzyme responsiveness. Using a rat MI model, we observed that these NPs were capable of successfully extravasating into the infarcted region of the heart where they were retained due to the active, enzyme-mediated targeting, remaining detectable after 1 week post administration without increasing macrophage recruitment. Furthermore, in vitro studies show that these NPs demonstrated successful drug release following MMP treatment and maintained drug bioactivity as evidenced by comparable MMP inhibition to free MMPi. This work establishes a targeted NP platform for delivering small-molecule therapeutics to the heart after MI, opening possibilities for myocardial infarction treatment.

Histone Deacetylase 3‐Directed PROTACs Have Anti‐inflammatory Potential by Blocking Polarization of M0‐like into M1‐like Macrophages

AbstractMacrophage polarization plays a crucial role in inflammatory processes. The histone deacetylase 3 (HDAC3) has a deacetylase‐independent function that can activate pro‐inflammatory gene expression in lipopolysaccharide‐stimulated M1‐like macrophages and cannot be blocked by traditional small‐molecule HDAC3 inhibitors. Here we employed the proteolysis targeting chimera (PROTAC) technology to target the deacetylase‐independent function of HDAC3. We developed a potent and selective HDAC3‐directed PROTAC, P7, which induces nearly complete HDAC3 degradation at low micromolar concentrations in both THP‐1 cells and human primary macrophages. P7 increases the anti‐inflammatory cytokine secretion in THP‐1‐derived M1‐like macrophages. Importantly, P7 decreases the secretion of pro‐inflammatory cytokines in M1‐like macrophages derived from human primary macrophages. This can be explained by the observed inhibition of macrophage polarization from M0‐like into M1‐like macrophage. In conclusion, we demonstrate that the HDAC3‐directed PROTAC P7 has anti‐inflammatory activity and blocks macrophage polarization, demonstrating that this molecular mechanism can be targeted with small molecule therapeutics.

Histone Deacetylase 3-Directed PROTACs Have Anti-inflammatory Potential by Blocking Polarization of M0-like into M1-like Macrophages.

Macrophage polarization plays a crucial role in inflammatory processes. The histone deacetylase 3 (HDAC3) has a deacetylase-independent function that can activate pro-inflammatory gene expression in lipopolysaccharide-stimulated M1-like macrophages and cannot be blocked by traditional small-molecule HDAC3 inhibitors. Here we employed the proteolysis targeting chimera (PROTAC) technology to target the deacetylase-independent function of HDAC3. We developed a potent and selective HDAC3-directed PROTAC, P7, which induces nearly complete HDAC3 degradation at low micromolar concentrations in both THP-1 cells and human primary macrophages. P7 increases the anti-inflammatory cytokine secretion in THP-1-derived M1-like macrophages. Importantly, P7 decreases the secretion of pro-inflammatory cytokines in M1-like macrophages derived from human primary macrophages. This can be explained by the observed inhibition of macrophage polarization from M0-like into M1-like macrophage. In conclusion, we demonstrate that the HDAC3-directed PROTAC P7 has anti-inflammatory activity and blocks macrophage polarization, demonstrating that this molecular mechanism can be targeted with small molecule therapeutics.

Pharmacokinetics and Pharmacodynamics of GalNAc-Conjugated siRNAs.

Small interfering RNAs (siRNAs) represent a new class of drugs with tremendous potential for battling previously "undruggable" diseases. After nearly 2 decades of efforts in addressing the problems of the poor drug profile of naked unmodified siRNAs, this new modality has finally come to fruition, with 5 agents (patisiran, givosiran, lumasiran, inclisiran, and vutrisiran) being approved since 2018, and with many others in the different phases of clinical development. Unlike small-molecule drugs and protein therapeutics, siRNAs have different sizes, distinct mechanisms of action, differing physicochemical and pharmacological properties, and, accordingly, a unique pharmacokinetic/pharmacodynamic (PK/PD) relationship. To support the continuous development of siRNAs, it is important to have a thorough and deep understanding of the PK/PD and clinical pharmacology related features of siRNAs. As most of the current siRNA products are conjugated by N-acetylgalactosamine (GalNAc), this review focuses on the PK/PD relationships and clinical pharmacology of GalNAc-conjugated siRNAs, including their absorption, distribution, metabolism, excretion (ADME) properties, PK/PD models, drug-drug interactions, clinical pharmacology in special populations, and safety evaluation. In addition, necessary background information related to the development of siRNAs as a therapeutic modality, including the mechanisms of action, the advantages of siRNAs, the problems of naked siRNAs, as well as the strategies used to enhance the clinical utility of siRNAs, have also been covered. The goal of this review is to serve as a "primer" on siRNA PK/PD, and I hope the readers, especially those who have a limited background on siRNA therapeutics, will have a fundamental understanding of siRNA PK/PD and clinical pharmacology after reading this review.

N-Acetylcysteine and Probenecid Adjuvant Therapy for Traumatic Brain Injury.

N-Acetylcysteine (NAC) has shown promise as a putative neurotherapeutic for traumatic brain injury (TBI). Yet, many such promising compounds have limited ability to cross the blood-brain barrier (BBB), achieve therapeutic concentrations in brain, demonstrate target engagement, among other things, that have hampered successful translation. A pharmacologic strategy for overcoming poor BBB permeability and/or efflux out of the brain of organic acid-based, small molecule therapeutics such as NAC is co-administration with a targeted or nonselective membrane transporter inhibitor. Probenecid is a classic ATP-binding cassette and solute carrier inhibitor that blocks transport of organic acids, including NAC. Accordingly, combination therapy using probenecid as an adjuvant with NAC represents a logical neurotherapeutic strategy for treatment of TBI (and other CNS diseases). We have completed a proof-of-concept pilot study using this drug combination in children with severe TBI-the Pro-NAC Trial (ClinicalTrials.gov NCT01322009). In this review, we will discuss the background and rationale for combination therapy with probenecid and NAC in TBI, providing justification for further clinical investigation.

Identification of CXCR4 inhibitors as a key therapeutic small molecule in renal fibrosis

The final stage of almost all chronic kidney diseases is renal fibrosis. Simple wounds or persistent inflammation can cause tissue inflammation, which, in the case of the kidney, results in scarring. Vascular sclerosis, tubulointerstitial fibrosis and glomerular fibrosis are all types of kidney fibrosis. Renal damage and fibrosis are caused by elevated expression of CXCR4. This study aimed to identify possible pharmacological agents which could bind to and inhibit isoform I of CXCR4 and determine their strength of interactions. The I-TASSER, Galaxyweb and Robetta were used to predict and refine the structure of the CXCR4 protein. ModBase was used to improve the loops, and then the quality was evaluated by using the ERRAT value (92.15) and Ramachandran plot. The improved 3D structure was subjected to small molecule database docking using Maestro (from Schrodinger) and the glide module. GROMACS was used to simulate molecules with the three top low glide scores and the best ADME properties. The best glide score was achieved by ligand ID 4990 (-11.5). Simulations, free energy landscape and residue decomposition analysis revealed that 4990 interacted more consistently with CXCR4 than the other two small molecules. Communicated by Ramaswamy H. Sarma

Ex-vivo drug screening of surgically resected glioma stem cells to replace murine avatars and provide personalise cancer therapy for glioblastoma patients.

With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas.

Modulated Fibrosis and Mechanosensing of Fibroblasts by SB525334 in Pediatric Subglottic Stenosis.

Subglottic stenosis (SGS) may result from prolonged intubation where fibrotic scar tissue narrows the airway. The scar forms by differentiated myofibroblasts secreting excessive extracellular matrix (ECM). TGF-β1 is widely accepted as a regulator of fibrosis; however, it is unclear how biomechanical pathways co-regulate fibrosis. Therefore, we phenotyped fibroblasts from pediatric patients with SGS to explore how key signaling pathways, TGF-β and Hippo, impact scarring and assess the impact of inhibiting these pathways with potential therapeutic small molecules SB525334 and DRD1 agonist dihydrexidine hydrochloride (DHX). Laryngeal fibroblasts isolated from subglottic as well as distal control biopsies of patients with evolving and maturing subglottic stenosis were assessed by α-smooth muscle actin immunostaining and gene expression for α-SMA, FN, HGF, and CTGF markers. TGF-β and Hippo signaling pathways were modulated during TGF-β1-induced fibrosis using the inhibitor SB525334 or DHX and analyzed by RT-qPCR for differential gene expression and atomic force microscopy for ECM stiffness. SGS fibroblasts exhibited higher α-SMA staining and greater inflammatory cytokine and fibrotic marker expression upon TGF-β1 stimulation (p < 0.05). SB525334 restored levels to baseline by reducing SMAD2/3 nuclear translocation (p < 0.0001) and pro-fibrotic gene expression (p < 0.05). ECM stiffness of stenotic fibroblasts was greater than healthy fibroblasts and was restored to baseline by Hippo pathway modulation using SB525334 and DHX (p < 0.01). We demonstrate that distinct fibroblast phenotypes from diseased and healthy regions of pediatric SGS patients respond differently to TGF-β1 stimulation, and SB525334 has the superior potential for subglottic stenosis treatment by simultaneously modulating TGF-β and Hippo signaling pathways. NA Laryngoscope, 134:287-296, 2024.

Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling.

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMTs) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukemia cells were treated with losmapimod, a known inhibitor of MAPK14 (p38α). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable of TMT-DDA in their ability to detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labeled quantitation in the TPP pipeline.

Selective Hydroxylation of C(sp3 )-H Bonds in Steroids.

Steroids are highly prevalent structures in small-molecule therapeutics, with the level of oxidation being key to their biological activity and physicochemical properties. These C(sp3 )-rich tetracycles contain many stereocentres, which are important for creating specific vectors and protein binding orientations. Therefore, the ability to hydroxylate steroids with a high degree of regio-, chemo- and stereoselectivity is essential for researchers working in this field. This review will cover three main methods for the hydroxylation of steroidal C(sp3 )-H bonds: biocatalysis, metal-catalysed C-H hydroxylation and organic oxidants, such as dioxiranes and oxaziridines.

Bicyclic peptides: Paving the road for therapeutics of the future

AbstractBicyclic peptides have emerged as one of the driving forces within the constrained peptide family. Due to their unique pharmaceutical attributes, peptide bicycles have garnered a reputation as promising therapeutics of the future. Combining advantages of small molecule therapeutics and biologics, the target binding qualities of this highly constrained class of molecules have been likened to those of antibodies. Over the past 5 years, the field of bicyclic peptides has advanced significantly. New bicyclic secondary peptide metabolites have been discovered in aquatic organisms and, importantly, a diverse assortment of bicyclic peptides has been synthesized de novo, exploring new chemical scaffolds. Biocompatible synthetic strategies that are suitable for large library screening platforms have helped to progress bicyclic peptides into clinical trials. This review aims to provide a comprehensive overview of the most recent progress in the field.

Solubility-Permeability Interplay in Facilitating the Prediction of Drug Disposition Routes, Extent of Absorption, Food Effects, Brain Penetration and Drug Induced Liver Injury Potential

Here I detail the use of measures of permeability rate and solubility in predicting drug disposition characteristics through the utilization of the Biopharmaceutics Drug Disposition Classification System (BDDCS) and the Extended Clearance Classification System (ECCS) as well as the accuracy of the systems in predicting the major route of elimination and the extent of oral absorption of a new small molecule therapeutics. I compare the BDDCS and ECCS with the FDA Biopharmaceutics Classification System (BCS). I also detail the use of the BCS in predicting food effects and the BDDCS in predicting brain disposition of small molecule therapeutics and in validating DILI predictive metrics. This review provides an update of the current status of these classification systems and their uses in the drug development process.

Complement therapeutics are coming of age in rheumatology.

The complement system was described over 100 years ago, and it is well established that activation of this pathway accompanies the great majority of autoimmune and inflammatory diseases. In addition, over three decades of work in murine models of human disease have nearly universally demonstrated that complement activation is upstream of tissue injury and the engagement of pro-inflammatory mechanisms such as the elaboration of cytokines and chemokines, as well as myeloid cell recruitment and activation. With that background, and taking advantage of advances in the development of biologic and small-molecule therapeutics, the creation and clinical evaluation of complement therapeutics is now rapidly expanding. This article reviews the current state of the complement therapeutics field, with a focus on their use in diseases cared for or consulted upon by rheumatologists. Included is an overview of the activation mechanisms and components of the system, in addition to the mechanisms by which the complement system interacts with other immune system constituents. The various therapeutic approaches to modulating the system in rheumatic and autoimmune diseases are reviewed. To understand how best to clinically assess the complement system, methods of its evaluation are described. Finally, next-generation therapeutic and diagnostic advances that can be envisioned for the future are discussed.

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are related RNA viruses responsible for severe respiratory infections and resulting disease in infants, elderly, and immunocompromised adults1–3. Therapeutic small molecule inhibitors that bind to the RSV polymerase and inhibit viral replication are being developed, but their binding sites and molecular mechanisms of action remain largely unknown4. Here we report a conserved allosteric inhibitory site identified on the L polymerase proteins of RSV and HMPV that can be targeted by a dual-specificity, non-nucleoside inhibitor, termed MRK-1. Cryo-EM structures of the inhibitor in complexes with truncated RSV and full-length HMPV polymerase proteins provide a structural understanding of how MRK-1 is active against both viruses. Functional analyses indicate that MRK-1 inhibits conformational changes necessary for the polymerase to engage in RNA synthesis initiation and to transition into an elongation mode. Competition studies reveal that the MRK-1 binding pocket is distinct from that of a capping inhibitor with an overlapping resistance profile, suggesting that the polymerase conformation bound by MRK-1 may be distinct from that involved in mRNA capping. These findings should facilitate optimization of dual RSV and HMPV replication inhibitors and provide insights into the molecular mechanisms underlying their polymerase activities.