Future Medicinal Chemistry Research Articles

From Serendipity to Rational Identification of the 5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one Core as a New Chemotype of AKT1 Inhibitors for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy whose prognosis is globally poor. In more than 60% of AML patients, the PI3K/AKTs/mTOR signaling pathway is aberrantly activated because of oncogenic driver alterations and further enhanced by chemotherapy as a mechanism of drug resistance. Against this backdrop, very recently we have started a multidisciplinary research project focused on AKT1 as a pharmacological target to identify novel anti-AML agents. Indeed, the serendipitous finding of the in-house compound T187 as an AKT1 inhibitor has paved the way to the rational identification of new active small molecules, among which T126 has emerged as the most interesting compound with IC50 = 1.99 ± 0.11 μM, ligand efficiency of 0.35, and a clear effect at low micromolar concentrations on growth inhibition and induction of apoptosis in AML cells. The collected results together with preliminary SAR data strongly indicate that the 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one derivative T126 is worthy of future biological experiments and medicinal chemistry efforts aimed at developing a novel chemical class of AKT1 inhibitors as anti-AML agents.

Structure–Activity Relationships of a Negative Allosteric Modulator of the D3 Dopamine Receptor and Investigation of its Binding Site

Dopamine receptors (DARs) are G protein‐coupled receptors (GPCRs) that regulate diverse physiological functions and are involved in the treatment and/or etiology of many neuropsychiatric disorders including schizophrenia and substance use disorder (SUD). DARs are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological profiles. Antagonists of D2‐like DARs are currently used in the therapies for many neuropsychiatric disorders. D3R‐selective antagonists have the potential to be better therapeutics for schizophrenia or SUD as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R‐preferring antagonists. Unfortunately, discovery of subtype‐selective compounds for the D3R and D2R has been challenging due to high sequence homology within the orthosteric binding sites of DARs. However, compounds that modulate receptor activities through interactions with less conserved allosteric sites have the potential to be highly selective. To find highly selective allosteric antagonists of the D3R, we screened the NIH Molecular Libraries Program 400,000+ small molecule library with a D3R‐mediated β‐arrestin recruitment assay. We found that one compound, MLS6357, was selective for the D3R over the D2R and D4R in several functional outputs including β‐arrestin recruitment and G‐protein activation. Radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross‐reactivity with other GPCRs. Additionally, Schild‐type functional assays showed that MLS6357 acts as a purely non‐competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized >60 analogs of MLS6357 using iterative medicinal chemistry approaches, which revealed structure–activity relationships and enabled further optimization of the scaffold. These efforts produced analogs that are 10‐fold and 30‐fold more potent than the parent compound in D3R‐mediated β‐arrestin recruitment and G‐protein activation assays, respectively. Moreover, some analogs appear to display functional selectivity for inhibition of G‐protein activation versus inhibition of β‐arrestin recruitment, and vice versa, and some also display inverse agonist activity. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R‐related pathophysiology.

Acute Neurotoxicity of Antisense Oligonucleotides After Intracerebroventricular Injection Into Mouse Brain Can Be Predicted from Sequence Features.

Antisense oligonucleotides are a relatively new therapeutic modality and safety evaluation is still a developing area of research. We have observed that some oligonucleotides can produce acute, nonhybridization dependent, neurobehavioral side effects after intracerebroventricular (ICV) dosing in mice. In this study, we use a combination of in vitro, in vivo, and bioinformatics approaches to identify a sequence design algorithm, which can reduce the number of acutely toxic molecules synthesized and tested in mice. We find a cellular assay measuring spontaneous calcium oscillations in neuronal cells can predict the behavioral side effects after ICV dosing, and may provide a mechanistic explanation for these observations. We identify sequence features that are overrepresented or underrepresented among oligonucleotides causing these reductions in calcium oscillations. A weighted linear combination of the five most informative sequence features predicts the outcome of ICV dosing with >80% accuracy. From this, we develop a bioinformatics tool that allows oligonucleotide designs with acceptable acute neurotoxic potential to be identified, thereby reducing the number of toxic molecules entering drug discovery pipelines. The informative sequence features we identified also suggest areas in which to focus future medicinal chemistry efforts.

Special Focus Issue - Targeted protein degradation: a new paradigm in medicinal chemistry.

Special Focus Issue - Targeted protein degradation: a new paradigm in medicinal chemistry.

KRAS(G12D) can be targeted by potent inhibitors via formation of salt bridge

KRAS mutation occurs in nearly 30% of human cancers, yet the most prevalent and oncogenic KRAS(G12D) variant still lacks inhibitors. Herein, we designed a series of potent inhibitors that can form a salt bridge with KRAS’s Asp12 residue. Our ITC results show that these inhibitors have similar binding affinity with both GDP-bound and GTP-bound KRAS(G12D), and our crystallographic studies reveal the structural basis of inhibitor binding-induced switch-II pocket in KRAS(G12D), experimentally confirming the formation of a salt bridge between the piperazine moiety of the inhibitors and the Asp12 residue of the mutant protein. Among KRAS family proteins and mutants, both ITC and enzymatic assays demonstrate the selectivity of the inhibitors for KRAS(G12D); and the inhibitors disrupt the KRAS–CRAF interaction. We also observed the inhibition of cancer cell proliferation as well as MAPK signaling by a representative inhibitor (TH-Z835). However, since the inhibition was not fully dependent on KRAS mutation status, it is possible that our inhibitors may have off-target effects via targeting non-KRAS small GTPases. Experiments with mouse xenograft models of pancreatic cancer showed that TH-Z835 significantly reduced tumor volume and synergized with an anti-PD-1 antibody. Collectively, our study demonstrates proof-of-concept for a strategy based on salt-bridge and induced-fit pocket formation for KRAS(G12D) targeting, which warrants future medicinal chemistry efforts for optimal efficacy and minimized off-target effects.

Mycobacterium abscessus drug discovery using machine learning

The prevalence of infections by nontuberculous mycobacteria is increasing, having surpassed tuberculosis in the United States and much of the developed world. Nontuberculous mycobacteria occur naturally in the environment and are a significant problem for patients with underlying lung diseases such as bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis. Current treatment regimens are lengthy, complicated, toxic and they are often unsuccessful as seen by disease recurrence. Mycobacterium abscessus is one of the most commonly encountered organisms in nontuberculous mycobacteria disease and it is the most difficult to eradicate. There is currently no systematically proven regimen that is effective for treating M. abscessus infections. Our approach to drug discovery integrates machine learning, medicinal chemistry and in vitro testing and has been previously applied to Mycobacterium tuberculosis. We have now identified several novel 1-(phenylsulfonyl)-1H-benzimidazol-2-amines that have weak activity on M. abscessus in vitro but may represent a starting point for future further medicinal chemistry optimization. We also address limitations still to be overcome with the machine learning approach for M. abscessus.

Trends of antimalarial marine natural products: progresses, challenges and opportunities.

Covering 1972 to 2021Malaria remains a significant public health problem in some regions of the world. The great efforts to control malaria have been severely compromised due to the widespread resistance of Plasmodium falciparum to nearly all frontline drugs. Pursuit of novel molecules from the sea will potentially result in new interventions against malaria, which are urgently needed to combat the increase of resistance. Focusing on the strategy of the "Blue Drug Bank", the molecules highlighted here can serve as an inspiration for future medicinal chemistry campaigns. This review covers the developments in the field of antimalarial marine lead compounds reported between 1972 and July 2021, and offers a comprehensive overview on their progresses and potentials. We selected 60 representative potential candidate molecules from 361 marine natural products, and highlighted their structure-activity relationships, molecular mechanisms of targets, and drug-like properties in order to assess their full potential to be developed. We summarized 107 clinically proven or potential antimalarial targets and their subcellular locations in the relevant target proteins, which linked the molecules to the target proteins at the subcellular level. Hence, it could be expected that natural products targeting different mechanisms may prove to be an effective strategy in antimalarial drug research and development in the future.

Broad-Spectrum Anticancer Activity and Pharmacokinetic Properties of a Prenyloxy-Substituted Indeno[1,2-b]indole Derivative, Discovered as CK2 Inhibitor.

Protein kinase CK2 is involved in regulating cellular processes, such as cell cycle, proliferation, migration, and apoptosis, making it an attractive anticancer target. We previously described a prenyloxy-substituted indeno[1,2-b]indole (5-isopropyl-4-(3-methylbut-2-enyloxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4p)) as a very potent inhibitor of CK2 holoenzyme (IC50 = 25 nM). Here, we report the broad-spectrum anticancer activity of 4p and provide substantial progress on its pharmacokinetic properties. Using a cell-based CK2 activity assay and live-cell imaging of cultured A431, A549, and LNCaP cancer cell lines, cellular CK2 target engagement was shown as well as strong antiproliferative, anti-migratory and apoptosis-inducing effects of 4p. Furthermore, evidence was found for the ability of 4p to disrupt A549 spheroid cohesion. A series of LC-MS/MS experiments revealed high and rapid cellular uptake (intracellular concentration is approximately 5 µM after 1 h incubation) and low metabolic stability of 4p. These results point to the value of 4p as a potent CK2 inhibitor with promising anticancer activities and should trigger future medicinal chemistry efforts to improve the drug-like properties of this compound.

The Medicinal Chemistry of Caffeine.

The purine alkaloid caffeine is the most widely consumed psychostimulant drug in the world and has multiple beneficial pharmacological activities, for example, in neurodegenerative diseases. However, despite being an extensively studied bioactive natural product, the mechanistic understanding of caffeine's pharmacological effects is incomplete. While several molecular targets of caffeine such as adenosine receptors and phosphodiesterases have been known for decades and inspired numerous medicinal chemistry programs, new protein interactions of the xanthine are continuously discovered providing potentially improved pharmacological understanding and a molecular basis for future medicinal chemistry. In this Perspective, we gather knowledge on the confirmed protein interactions, structure activity relationship, and chemical biology of caffeine on well-known and upcoming targets. The diversity of caffeine's molecular activities on receptors and enzymes, many of which are abundant in the CNS, indicates a complex interplay of several mechanisms contributing to neuroprotective effects and highlights new targets as attractive subjects for drug discovery.

Importance of Biometals as Targets in Medicinal Chemistry: An Overview about the Role of Zinc (II) Chelating Agents

Zinc (II) is an important biometal in human physiology. Moreover, in the last two decades, it was deeply studied for its involvement in several pathological states. In particular, the regulation of its concentration in synaptic clefts can be fundamental for the treatment of neurodegenerative diseases, such as Alzheimer’s disease (AD). Zinc (II) is also a constituent of metalloenzymes (i.e., matrix metalloproteinases, MMPs, and carbonic anhydrases, CAs) with catalytic function; therefore, it can be an important target for the inhibition of these proteins, frequently involved in cancer onset. This review is focused on the significance of zinc (II) chelating agents in past and future medicinal chemistry research, and on the importance of selectivity in order to revamp the possibility of their use in therapy, often hindered by possible side effects.

Methodology for the identification of small molecule inhibitors of the Fanconi Anaemia ubiquitin E3 ligase complex

DNA inter-strand crosslinks (ICLs) threaten genomic stability by creating a physical barrier to DNA replication and transcription. ICLs can be caused by endogenous reactive metabolites or from chemotherapeutics. ICL repair in humans depends heavily on the Fanconi Anaemia (FA) pathway. A key signalling step of the FA pathway is the mono-ubiquitination of Fanconi Anaemia Complementation Group D2 (FANCD2), which is achieved by the multi-subunit E3 ligase complex. FANCD2 mono-ubiquitination leads to the recruitment of DNA repair proteins to the site of the ICL. The loss of FANCD2 mono-ubiquitination is a common clinical feature of FA patient cells. Therefore, molecules that restore FANCD2 mono-ubiquitination could lead to a potential drug for the management of FA. On the other hand, in some cancers, FANCD2 mono-ubiquitination has been shown to be essential for cell survival. Therefore, inhibition of FANCD2 mono-ubiquitination represents a possible therapeutic strategy for cancer specific killing. We transferred an 11-protein FANCD2 mono-ubiquitination assay to a high-throughput format. We screened 9,067 compounds for both activation and inhibition of the E3 ligase complex. The use of orthogonal assays revealed that candidate compounds acted via non-specific mechanisms. However, our high-throughput biochemical assays demonstrate the feasibility of using sophisticated and robust biochemistry to screen for small molecules that modulate a key step in the FA pathway. The future identification of FA pathway modulators is anticipated to guide future medicinal chemistry projects with drug leads for human disease.

Structure‐Activity Relationships of a Negative Allosteric Modulator of the D3 Dopamine Receptor and Investigation of its Binding Site

Dopamine receptors (DARs) are G protein‐coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward‐related behaviors, and are involved in the treatment or etiology of many neuropsychiatric disorders including schizophrenia, substance use disorder (SUD), and Parkinson’s disease. DARs are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. Antagonists of D2‐like DARs are currently used as drug therapies for many psychiatric disorders. While the D2R is highly expressed in the dorsal striatum, which regulates movement, the D3R has a more limited distribution to limbic regions associated with the control of mood and emotion. D3R‐selective antagonists may therefore be useful as schizophrenia or SUD therapeutics as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R‐preferring antagonists. Discovery of subtype selective compounds for the D3R and D2R has been challenging due to high sequence homology within their orthosteric binding sites. However, compounds that modulate receptor activity through interactions with a less conserved allosteric site of the receptor have the potential to be highly selective. To find highly selective allosteric antagonists of the D3R, our lab utilized a high‐throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library using a D3R‐mediated β‐arrestin recruitment assay. We found one compound, MLS6357, that was selective for the D3R versus the D2R in several functional outputs including β‐arrestin recruitment and G‐protein activation. Further, radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross‐reactivity with other GPCRs. Additionally, Schild‐type functional assays found that MLS6357 is a purely non‐competitive negative allosteric modulator of the D3R. We synthesized 46 analogs of MLS6357 using iterative medicinal chemistry and tested them for activity which revealed structure activity‐relationships and enabled further refinement of the scaffold. These efforts produced modulators that are 5‐ to 9‐fold more potent than the original hit compound. To identify the allosteric site on the D3R, we utilized D3R/D2R chimeras which revealed receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R‐related pathophysiology.Support or Funding InformationNINDS Intramural Research Program

Welcome to Volume 12 of Future Medicinal Chemistry.

Welcome to Volume 12 of Future Medicinal Chemistry.

Scaffold Simplification Strategy Leads to a Novel Generation of Dual Human Immunodeficiency Virus and Enterovirus-A71 Entry Inhibitors.

Currently, there are only three FDA-approved drugs that inhibit human immunodeficiency virus (HIV) entry-fusion into host cells. The situation is even worse for enterovirus EV71 infection for which no antiviral therapies are available. We describe here the discovery of potent entry dual inhibitors of HIV and EV71. These compounds contain in their structure three or four tryptophan (Trp) residues linked to a central scaffold. Critical for anti-HIV/EV71 activity is the presence of extra phenyl rings, bearing one or two carboxylates, at the C2 position of the indole ring of each Trp residue. The most potent derivatives, 22 and 30, inhibit early steps of the replicative cycles of HIV-1 and EV-A71 by interacting with their respective viral surfaces (glycoprotein gp120 of HIV and the fivefold axis of the EV-A71 capsid). The high potency, low toxicity, facile chemical synthesis, and great opportunities for chemical optimization make them useful prototypes for future medicinal chemistry studies.

Synthesis of Bioderived Cinnolines and Their Flow-Based Conversion into 1,4-Dihydrocinnoline Derivatives

Starting from phenylhydrazine and glucose, a versatile cinnoline scaffold was obtained on a multigram scale and further derivatized. A simple continuous-flow hydrogenation process permits the conversion of selected cinnolines into their 1,4-dihydrocinnoline counterparts. These products are generated in high yields and high purities with residence times of less than one minute and, along with their cinnoline precursors, are expected to serve as valuable heterocyclic building blocks for future medicinal chemistry programs.

Identification of Fromiamycalin and Halaminol A from Australian Marine Sponge Extracts with Anthelmintic Activity against Haemonchus contortus.

There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus — a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata, a known pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC50) = 26.6 ± 0.74 µM) and L4 motility (IC50 = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC50 ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.

The Anti-mycobacterial Activity of a Diterpenoid-Like Molecule Operates Through Nitrogen and Amino Acid Starvation.

A library of 14 minimally cytotoxic diterpenoid-like compounds (CC50 > 70 μM on HepG2 human liver cells) was screened against Mycobacterium smegmatis, Staphylococcus aureus, and Escherichia coli to determine antimicrobial activity. Some compounds with a phenethyl alcohol (PE) core substituted with a β-cyclocitral derivative demonstrated anti-mycobacterial activity, with the most active being compound 1 (MIC = 23.4 mg/L, IC50 = 0.6 mg/L). Lower activity was exhibited against S. aureus, while no activity was displayed against E. coli. Low cytotoxicity was re-confirmed on HepG2 cells and additionally on RAW 264.7 murine macrophages (SI for both cell lines > 38). The sub-lethal (IC50 at 6 h) effect of compound 1 on M. smegmatis was examined through untargeted metabolomics and compared to untreated bacteria and bacteria treated with sub-lethal (IC50 at 6 h) concentrations of the antituberculosis drugs ethambutol, isoniazid, kanamycin, and streptomycin. The study revealed that compound 1 acts differently from the reference antibiotics and that it significantly affects amino acid, nitrogen, nucleotides and folate-dependent one-carbon metabolism of M. smegmatis, giving some insights about the mode of action of this molecule. A future medicinal chemistry optimization of this new anti-mycobacterial core could lead to more potent molecules.

Identification of a novel scaffold for a small molecule GPR139 receptor agonist

GPR139 is an orphan G protein-coupled receptor (GPCR) that is primarily expressed in the brain in regions known to regulate motor control and metabolism. Here, we screened a diverse 4,000 compound library in order to identify GPR139 agonists. We identified 11 initial hits in a calcium mobilization screen, including one compound, AC4, which contains a different chemical scaffold to what has previously been described for GPR139 agonists. Our mutagenesis data shows that AC4 interacts with the same hotspots in the binding site of GPR139 as those reported to interact with the reference agonists 1a and 7c. We additionally tested and validated 160 analogs in a calcium mobilization assay and found 5 compounds with improved potency compared to AC4. In total, we identified 36 GPR139 agonists with potencies in the nanomolar range (90–990 nM). The most potent compounds were confirmed as GPR139 agonists using an orthogonal ERK phosphorylation assay where they displayed a similar rank order of potency. Accordingly, we herein introduce multiple novel GPR139 agonists, including one with a novel chemical scaffold, which can be used as tools for future pharmacological and medicinal chemistry exploration of GPR139.

Computer-Assisted Discovery and Structural Optimization of a Novel Retinoid X Receptor Agonist Chemotype.

As universal heterodimer partners of many nuclear receptors, the retinoid X receptors (RXRs) constitute key transcription factors. They regulate cell proliferation, differentiation, inflammation, and metabolic homeostasis and have recently been proposed as potential drug targets for neurodegenerative and inflammatory diseases. Owing to the hydrophobic nature of RXR ligand binding sites, available synthetic RXR ligands are lipophilic, and their structural diversity is limited. Here, we disclose the computer-assisted discovery of a novel RXR agonist chemotype and its systematic optimization toward potent RXR modulators. We have developed a nanomolar RXR agonist with high selectivity among nuclear receptors and superior physicochemical properties compared to classical rexinoids that appears suitable for in vivo applications and as lead for future RXR-targeting medicinal chemistry.

Structural Basis of Substrate Recognition and Covalent Inhibition of Cdu1 from Chlamydia trachomatis.

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionarily related protease adenain, a target-hopping screening approach on a focused set of adenain inhibitors was investigated. The cyanopyrimidine-based inhibitors identified represent the first active-site-directed small-molecule inhibitors of Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyanopyrimidines, as well as with its substrate ubiquitin, were obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into the substrate recognition of Cdu1, active-site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong basis for future structure-guided medicinal chemistry optimization of this cyanopyrimidine scaffold into more potent and selective Cdu1 inhibitors.