Development of a New Class of Inhibitors for the Malarial Aspartic Protease Plasmepsin II Based on a Central 7‐Azabicyclo[2.2.1]heptane Scaffold

Plasmepsin II (PMII), a malarial aspartic protease involved in the catabolism of hemoglobin in parasites of the genus Plasmodium, and renin, a human aspartic protease, share 35% sequence identity in their mature chains. Structures of 4‐arylpiperidine inhibitors complexed to human renin were reported by Roche recently. The major conformational changes, compared to a structure of renin, with a peptidomimetic inhibitor were identified and subsequently modeled in a structure of PMII (Fig. 1). This distorted structure of PMII served as active‐site model for a novel class of PMII inhibitors, according to a structure‐based de novo design approach (Fig. 2). These newly designed inhibitors feature a rigid 7‐azabicyclo[2.2.1]heptane scaffold, which, in its protonated form, is assumed to undergo ionic H‐bonding with the two catalytic Asp residues at the active site of PMII. Two substituents depart from the scaffold for occupancy of either the S1/S3 or S2′‐pocket and the hydrophobic flap pocket, newly created by the conformational changes in PMII. The inhibitors synthesized starting from N‐Boc‐protected 7‐azabicyclo[2.2.1]hept‐2‐ene ( 6 ; Schemes 1–5) displayed up to single‐digit micromolar activity (IC₅₀ values) toward PMII and good selectivity towards renin. The clear structure? activity relationship (SAR; Table) provides strong validation of the proposed conformational changes in PMII and the occupancy of the resulting hydrophobic flap pocket by our new inhibitors.     

Synthesis and evaluation of a new class of tertiary alcohol based BACE-1 inhibitors

BACE-1 has emerged as one of the best characterized targets for future Alzheimer therapy. In accordance with the successful identification of masked inhibitors of HIV-1 protease, we envisioned that tert-alcohol containing transition-state mimicking structures would also be worthwhile evaluating as BACE-1 inhibitors. Twelve novel inhibitors were prepared via synthetic routes using epoxyalcohol derivates as key intermediates. The best synthesized tert-hydroxy inhibitor exhibited a BACE-1 IC₅₀ value of 0.38 μM.     

New indolic non-peptidic HIV protease inhibitors from (S)-glycidol: synthesis and preliminary biological activity

A series of non-peptidic HIV protease inhibitors were synthesized starting from the same optically active precursor, (S)-glycidol. The substrate was easily converted into different indolic sulfonamides or amines by regioselective reactions. The preliminary inhibitory activity was evaluated.     

Development of Reduced Peptide Bond Pseudopeptide Michael Acceptors for the Treatment of Human African Trypanosomiasis

Human African Trypanosomiasis (HAT) is an endemic protozoan disease widespread in the sub-Saharan region that is caused by T. b. gambiense and T. b. rhodesiense. The development of molecules targeting rhodesain, the main cysteine protease of T. b. rhodesiense, has led to a panel of inhibitors endowed with micro/sub-micromolar activity towards the protozoa. However, whilst impressive binding affinity against rhodesain has been observed, the limited selectivity towards the target still remains a hard challenge for the development of antitrypanosomal agents. In this paper, we report the synthesis, biological evaluation, as well as docking studies of a series of reduced peptide bond pseudopeptide Michael acceptors (SPR10–SPR19) as potential anti-HAT agents. The new molecules show K_i values in the low-micro/sub-micromolar range against rhodesain, coupled with k_2nd values between 1314 and 6950 M⁻¹ min⁻¹. With a few exceptions, an appreciable selectivity over human cathepsin L was observed. In in vitro assays against T. b. brucei cultures, SPR16 and SPR18 exhibited single-digit micromolar activity against the protozoa, comparable to those reported for very potent rhodesain inhibitors, while no significant cytotoxicity up to 70 µM towards mammalian cells was observed. The discrepancy between rhodesain inhibition and the antitrypanosomal effect could suggest additional mechanisms of action. The biological characterization of peptide inhibitor SPR34 highlights the essential role played by the reduced bond for the antitrypanosomal effect. Overall, this series of molecules could represent the starting point for further investigations of reduced peptide bond-containing analogs as potential anti-HAT agents     

The Synthesis of Diphenyl Phosphonate Analogues of α-Amino Acids as Enzyme Inhibitors

Abstract

α-Aminoalkylphosphonic acids are analogues of natural aminoacids and as such have been the subject of much research effort over past years¹. The diphenyl esters of α- aminoalkylphosphonic acids are particularly potent and show high selectivity as irreversible inhibitors of serine proteinases. Thus far, α-aminoalkylphosphonic acid ester analogues of a number of aliphatic- and aromatic aminoacids have been prepared including valine, phenylalanine, tryptophan, and tyrosine², and the basic aminoacids ornithine, lysine. etc.³. We have now also prepared the a-diphenyl phosphonate analogues of the acidic aminoacids, aspartic and glutamic⁴. These have been examined as potential inactivators of serine proteinases exhibiting a P₁ specificity for aspartate and glutamate, e.g. S. aureus V8 protease and granzyme B.     

Structural and electronic properties of new fullerene derivatives and their possible application as HIV-1 protease inhibitors

Density functional theory (DFT) calculations have been carried out at the hybrid Becke 3-Lee–Yang–Parr; B3LYP/3-21G** level of theory to study two series of hydroxy-chalca-acetic acid-(4-pyrrolidin-1-yl-phenyl) ester [C₆₀-C₂H₄N-(4-XCOCH₂OH)C₆H₄] and hydroxy-chalcoacetic acid-[2-(2-hydroxy-acetylchalcanyl)-4-pyrrolidin-1-yl-phenyl] ester[C₆₀-C₂H₄N-(3,4-XCOCH₂OH)C₆H₄]. The X atom is O, S or Se for the two series. The vibrational spectra, physical, chemical, thermodynamics and Quantitative Structure Activity Relationship (QSAR) properties of the studied molecules are calculated and discussed. We have evaluated these molecules as HIV-1 protease inhibitors based on the hydrogenation interaction between the hydroxymethylcarbonyl (HMC) groups and the two aspartic acid of the HIV-1 protease active site. Results show that some of the investigated fullerene-based derivatives can be considered promising as HIV-1 protease inhibitors.     

New approach for natural products screening by real-time monitoring of hemoglobin hydrolysis using quartz crystal microbalance

The hemoglobin (Hb) released from erythrocytes is a primary nutritive component for many blood-feeding parasites. The aspartic protease cathepsin D is a hemoglobinase that is involved in the Hb degradation process and is considered an interesting target for chemotherapy intervention. However, traditional enzymatic assays for studying Hb degradation utilize spectrophotometric techniques, which do not allow real-time monitoring and can present serious interference problems. Herein, we describe a biosensor using simple approach for the real-time monitoring of Hb hydrolysis as well as an efficient screening method for natural products as enzymatic inhibitors using a quartz crystal microbalance (QCM) technique. Hemoglobin was anchored on the quartz crystal surface using mixed self-assembled monolayers. The addition of the enzyme caused a mass change (frequency shift) due to Hb hydrolysis, which was monitored in real time. From the frequency change patterns of the Hb-functionalized QCM, we evaluated the enzymatic reaction by determining the kinetic parameters of product formation (k_cat). The QCM enzymatic assay using immobilized human Hb was shown to be an excellent approach for screening possible inhibitors in complex mixtures, opening up a new avenue for the discovery of novel inhibitors.     

Synthesis of novel HIV-1 protease inhibitors based on carbohydrate scaffolds

The synthesis of peptidomimetic inhibitors of HIV-1 protease based on 6-deoxy-6-amino-β-d-glucopyranoside and 6-deoxy-6-amino-β-d-mannopyranoside scaffolds has been achieved. The inhibitors had IC₅₀ values in the micromolar range. The results provide a platform for the development of more potent carbohydrate based inhibitors of HIV-1 and other aspartic proteases.     

Computational systematic selectivity of the Fasalog inhibitors between ROCK-I and ROCK-II kinase isoforms in Alzheimer’s disease

Human Rho-associated coiled-coil forming kinase (ROCK) is a class of essential neurokinases that consists of two structurally conserved isoforms ROCK-I and ROCK-II; they have been revealed to play distinct roles in the pathogenesis of Alzheimer’s disease (AD) and other neurological disorders. Selective targeting of the two kinase isoforms with small-molecule inhibitors is a great challenge due to the surprisingly high homology in kinase domain (92 %) and the full identity in kinase active site (100 %). Here, we describe a computational protocol to systematically profile the selectivity of Fasudil and its 25 analogs (termed as Fasalogs) between the two kinase isoforms. It is suggested that the substitution of Fasudil’s 1,4-diazepane moiety with rigid ring such as Ripasudil and Dimehtylfasudil would render the resulting inhibitors of ROCK-II over ROCK-I (II-o-I) selectivity, while the substitution with long, flexible group such as H-89 and BDBM92607 tends to have I-o-II selectivity. Structural analysis reveals that the inhibitor affinity is not only determined by the identical active site, but also contributed from the non-identical first and second shells of the site as well as other non-conserved kinase regions, which can indirectly influence the active site and inhibitor binding through allosteric effect. A further kinase assay basically confirms the computational findings, which also exhibits a good consistence with theoretical selectivity over 10 tested samples (R_p = 0.89). In particular, the Fasalog compounds Dimehtylfasudil and H-89 are identified as II-o-I and I-o-II selective inhibitors. They can be considered as promising lead molecular entities to develop new specific ROCK isoform-selective Fasalog inhibitors.     

Non-peptidic natural products as ubiquitin-proteasome inhibitors

Approval of bortezomib has validated ubiquitin-proteasome pathway as an important target for treatment of haematological malignancies. However, clinical shortcomings of bortezomib, a covalent peptide proteasome inhibitor, has prompted a paradigm shift in anti-proteasome drug discovery towards development of non-peptidic inhibitors and targeting of upstream ubiquitin system which has drawn traction for interdisciplinary forays. It is being widely recognized that natural products provide valuable leads in the discovery of potent, chemically diverse, non-peptidic inhibitors of 20S proteasome and of key enzymes involved in ubiquitination machinery. As a result, total synthesis of natural, non-peptidic inhibitors of ubiquitin-proteasome pathway has emerged as a critical interlink between organic synthesis, medicinal chemistry, biochemical profiling and drug discovery. An up-to-date account of contextual synthetic challenges, strategies and accomplishments as well as mapping of the chemical diversity space around the natural scaffolds has been captured in this review.     

Improved cyclic urea inhibitors of the HIV-1 protease: synthesis,potency, resistance profile,human pharmacokinetics and X-ray crystal structure of DMP 450

Background: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds.Results: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg⁻¹ DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC₉₀ suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response.Conclusions: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.     

Novel anti-Plasmodial hits identified by virtual screening of the ZINC database

Increased resistance of Plasmodium falciparum to most available drugs challenges the control of malaria. Studies with protease inhibitors have suggested important roles for the falcipain family of cysteine proteases. These enzymes act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. In order to find potential new antimalarial drugs, we screened in silico the ZINC database using two different protocols involving structure- and ligand-based methodologies. Our search identified 19 novel low micromolar inhibitors of cultured chloroquine resistant P. falciparum. The most active compound presented an IC₅₀ value of 0.5 μM against cultured parasites and it also inhibited the cysteine protease falcipain-2 (IC₅₀ = 25.5 μM). These results identify novel classes of antimalarials that are structurally different from those currently in use and which can be further derivatized to deliver leads suitable for optimisation.     

Novel fullerene receptors based on calixarene-porphyrin conjugates

Several different synthetic approaches enabling a direct covalent connection between the meso-position of porphyrin and the upper rim of calix[4]arene have been studied. The best results were obtained via condensation of an excess of pyrrole and p-methylbenzaldehyde with calix[4]arene-5,17-dialdehyde under BF₃·Et₂O catalysis in CHCl₃. Subsequent oxidation of the intermediate porphyrinogen gave the corresponding bis-porphyrin-calixarene conjugate in 15% overall yield. The ¹H NMR complexation study revealed the pronounced selectivity of the bis-porphyrin derivative towards C₇₀ fullerene.     

Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (β5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the β5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure–activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.     

Combinatorial Chemistry Online - Volume 4, Issue 11, October 2002

Current literature highlights: Inhibitors of human rhinovirus 3C protease - The human rhinoviruses (HRVs) are members of the picornavirus family and are the single most causative agent of the common cold. Over 100 serotypes of the virus exist, so immunisation is an impractical approach to prevent the infection. Rhinoviruses contain a positive-sense strand of RNA that is translated to a large polyprotein in infected cells. This polyprotein is cleaved by viral proteases to yield mature viral enzymes and structural proteins. The 3C protease (3CP) does the majority of the proteolytic processing. Inhibition of this viral protease by a small molecule agent should stop viral replication and thus control the extent of infection. Small molecule inhibitors, such as isatins and homophthalimides, are known in the literature but these all suffer from problems such as cellular toxicity and have only modest antiviral activity. Low molecular weight non-peptidic HRV 3CP inhibitors have been synthesised (Structure-based design of a parallel synthetic array directed toward the discovery of irreversible inhibitors of human rhinovirus 3C protease, [1]).     

Potent Inhibitors of Malarial Aspartic Proteases,the Plasmepsins,by Hydroformylation of Substituted 7‐Azanorbornenes

The increasing prevalence of multidrug‐resistant strains of the malarial parasite Plasmodium falciparum requires the urgent development of new therapeutic agents with novel modes of action. The vacuolar malarial aspartic proteases plasmepsin (PM) I, II, and IV are involved in hemoglobin degradation and play a central role in the growth and maturation of the parasite in the human host. We report the structure‐based design, synthesis, and in vitro evaluation of a new generation of PM inhibitors featuring a highly decorated 7‐azabicyclo[2.2.1]heptane core. While this protonated central core addresses the catalytic Asp dyad, three substituents bind to the flap, the S1/S3, and the S1′ pockets of the enzymes. A hydroformylation reaction is the key synthetic step for the introduction of the new vector reaching into the S1′ pocket. The configuration of the racemic ligands was confirmed by extensive NMR and X‐ray crystallographic analysis. In vitro biological assays revealed high potency of the new inhibitors against the three plasmepsins (IC₅₀ values down to 6 nM ) and good selectivity towards the closely related human cathepsins D and E. The occupancy of the S1′ pocket makes an essential contribution to the gain in binding affinity and selectivity, which is particularly large in the case of the PM IV enzyme. Designing non‐peptidic ligands for PM II is a valid route to generate compounds that inhibit the entire family of vacuolar plasmepsins.     

Rational design of thioamide peptides as selective inhibitors of cysteine protease cathepsin L

Aberrant levels of cathepsin L (Cts L), a ubiquitously expressed endosomal cysteine protease, have been implicated in many diseases such as cancer and diabetes. Significantly, Cts L has been identified as a potential target for the treatment of COVID-19 due to its recently unveiled critical role in SARS-CoV-2 entry into the host cells. However, there are currently no clinically approved specific inhibitors of Cts L, as it is often challenging to obtain specificity against the many highly homologous cathepsin family cysteine proteases. Peptide-based agents are often promising protease inhibitors as they offer high selectivity and potency, but unfortunately are subject to degradation in vivo. Thioamide substitution, a single-atom O-to-S modification in the peptide backbone, has been shown to improve the proteolytic stability of peptides addressing this issue. Utilizing this approach, we demonstrate herein that good peptidyl substrates can be converted into sub-micromolar inhibitors of Cts L by a single thioamide substitution in the peptide backbone. We have designed and scanned several thioamide stabilized peptide scaffolds, in which one peptide, R^S_1A, was stabilized against proteolysis by all five cathepsins (Cts L, Cts V, Cts K, Cts S, and Cts B) while inhibiting Cts L with >25-fold specificity against the other cathepsins. We further showed that this stabilized R^S_1A peptide could inhibit Cts L in human liver carcinoma lysates (IC₅₀ = 19 μM). Our study demonstrates that one can rationally design a stabilized, specific peptidyl protease inhibitor by strategic placement of a thioamide and reaffirms the place of this single-atom modification in the toolbox of peptide-based rational drug design.

Information on the effects of sidechain and backbone modification on the activity of cathepsin (Cts) L, V, K, S, and B was used to design a thioamide peptide that is inert to all Cts and selectively inhibits Cts L.     

Fluorinated peptidomimetics: synthesis, conformational and biological features

Peptides modified with fluoroalkyl functions in key backbone positions have been scarcely studied so far. Thus, little is known about their synthesis, their structural and physico-chemical properties, and their biological features. Our interest in this field of research led to the development of stereocontrolled synthetic protocols, both in solution and in solid phase, for many different fluoroalkyl peptidomimetics, some of which are overviewed in this paper: (a) ψ[CH(CF₃)NH]-peptide mimics holding a great potential as hybrids between natural peptides and hydrolytic transition state analogs; (b) trifluoromethyl (Tfm) malic peptidomimetics as micromolar inhibitors of some matrix metalloproteinases; (c) bis-Tfm analogs of Pepstatin A, that are nanomolar and selective inhibitors of the protozoal aspartyl protease Plasmepsin II.     

Synthesis and biochemical characterisation of fluorinated analogues of pepstatin A and grassystatin A

Pepstatin A and grassystatin A are natural, statine-containing peptides that act as inhibitors of aspartic protease enzymes. In this work, stereoselective fluorination is investigated as a strategy for enhancing the pharmacodynamic and pharmacokinetic properties of these lead compounds. Fluorination is found to modestly affect the protease inhibitory potency, leading to the identification of two highly active new inhibitors of the cancer-associated protease, cathepsin D. However, no dramatic changes are observed in terms of target selectivity, lipophilicity, membrane permeability or metabolic stability.     

Computational design of novel cyclic urea as HIV-1 protease inhibitor

A series of novel cyclic urea molecules 5,6-dihydroxy-1,3-diazepane-2,4,7-trione as HIV-1 protease inhibitors were designed using computational techniques. The designed molecules were compared with the known cyclic urea molecules by performing docking studies, calculating their ADME (Absorption, Distribution, Metabolism, and Excretion) properties and protein ligand interaction energy. These novel molecules were designed by substituting the P ₁/P′ ₁ positions (4 ^th and 7 ^th position of 1, 3-diazepan-2-one) with double bonded oxygens. This reduces the molecular weight and increases the bioavailability, indicating better ADME properties. The docking studies showed good binding affinity towards HIV-1 protease. The biological activity of these inhibitors were predicted by a model equation generated by the regression analysis between biological activity (log 1/K ⁱ ) of known inhibitors and their protein ligand interaction energy. The synthetic studies are in progress.