P1 Position Research Articles

Substrate Peptidomimetic Inhibitors of P. falciparum Plasmepsin X with Potent Antimalarial Activity.

Plasmepsin X (PMX) is an aspartyl protease that processes proteins essential for Plasmodium parasites to invade and egress from host erythrocytes during the symptomatic asexual stage of malaria. PMX substrates possess a conserved cleavage region denoted by the consensus motif, SFhE (h=hydrophobic amino acid). Peptidomimetics reflecting the P3 -P1 positions of the consensus motif were designed and showed potent and selective inhibition of PMX. It was established that PMX prefers Phe in the P1 position, di-substitution at the β-carbon of the P2 moiety and a hydrophobic P3 group which was supported by modelling of the peptidomimetics in complex with PMX. The peptidomimetics were shown to arrest asexual P. falciparum parasites at the schizont stage by impairing PMX substrate processing. Overall, the peptidomimetics described will assist in further understanding PMX substrate specificity and have the potential to act as a template for future antimalarial design.

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

Evaluation of shape, size, and location of mental foramen in dentulous and edentulous among Saudi population using 3D cone-beam computed tomography

Background: Mental foramen (MF) and its accessories are the important anatomical considerations while placing implants or doing surgical procedures in and around the mandibular premolar region. This study aimed to evaluate the shape, size, and location of mental foramen in dentulous and edentulous patients among the Saudi population using 3D cone-beam computed tomography (CBCT). Methods: In this retrospective study, CBCT scans that were taken between 2015 and 2020 from Riyadh Elm University were retrieved. A total of 180 samples of CBCT (90 dentate and 90 edentulous) were analyzed. Prevalence of different horizontal positions of the mental foramen (P1 to P6) and other additive parameters like the distance from mental foramen to alveolar crest and inferior border of the mandible, along with the mental foramen angle was assessed. The difference in the mental foramen location among dentate and edentulous subjects was assessed. Gender and age variation also was assessed. All the data were statistically analyzed using SPSS. Results: The predominant horizontal position is P4 followed by P3 (59 % in males and 63 % in females at P4, and 15 % each in males and females at P3, respectively). The horizontal position of the mental foramen and gender showed a statistical significance difference, especially at the P3B, P5, and P4 positions. Moreover, a statistically significant difference was seen in the mental foramen to the mandibular inferior border of the mandible (MF_MSB) and the width of mental foramen in the transverse section (MFW). Comparison of the mental foramen among dentate and edentulous subjects showed a statistically significant difference. There was a change in the mental foramen with age. Conclusions: Based on the methodology and sample of this study, it can be concluded that the edentulism only reduced the dimension of the mental foramen opening.

Evaluating the Properties of Ginger Protease-Degraded Collagen Hydrolysate and Identifying the Cleavage Site of Ginger Protease by Using an Integrated Strategy and LC-MS Technology.

(1) Methods: An integrated strategy, including in vitro study (degree of hydrolysis (DH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) and in vivo study (absorption after oral administration in rats), was developed to evaluate the properties of the fish skin gelatin hydrolysates prepared using different proteases (pepsin, alkaline protease, bromelain, and ginger protease). Meanwhile, in order to identify the hydrolysis site of ginger protease, the peptides in the ginger protease-degraded collagen hydrolysate (GDCH) were comprehensively characterized by liquid chromatography/tandem mass spectrometry (LC-MS) method. (2) Results: The GDCH exhibited the highest DH (20.37%) and DPPH radical scavenging activity (77.73%), and in vivo experiments showed that the GDCH was more efficiently absorbed by the gastrointestinal tract. Further oral administration experiments revealed that GDCH was not entirely degraded to free amino acids and can be partially absorbed as dipeptides and tripeptides in intact forms, including Pro-Hyp, Gly-Pro-Hyp, and X-Hyp-Gly tripeptides. LC-MS results determined the unique substrate specificity of ginger protease recognizing Pro and Hyp at the P2 position based on the amino acids at the P2 position from the three types of tripeptides (Gly-Pro-Y, X-Hyp-Gly, and Z-Pro-Gly) and 136 identified peptides (>4 amino acids). Interestingly, it suggested that ginger protease can also recognize Ala in the P2 position. (3) Conclusions: This study comprehensively evaluated the properties of GDCH by combining in vitro and in vivo strategies, and is the first to identify the cleavage site of ginger protease by LC-MS technique. It provides support for the follow-up study on the commercial applications of ginger protease and bioactivities of the hydrolysate produced by ginger protease.

Structural plasticity in I-Ag7 links autoreactivity to hybrid insulin peptides in type I diabetes

We recently provided evidence for promiscuous recognition of several different hybrid insulin peptides (HIPs) by the highly diabetogenic, I-Ag7-restricted 4.1-T cell receptor (TCR). To understand the structural determinants of this phenomenon, we solved the structure of an agonistic HIP/I-Ag7 complex, both in isolation as well as bound to the 4.1-TCR. We find that HIP promiscuity of the 4.1-TCR is dictated, on the one hand, by an amino acid sequence pattern that ensures I-Ag7 binding and, on the other hand, by the presence of three acidic residues at positions P5, P7 and P8 that favor an optimal engagement by the 4.1-TCR’s complementary determining regions. Surprisingly, comparison of the TCR-bound and unbound HIP/I-Ag7 structures reveals that 4.1-TCR binding triggers several novel and unique structural motions in both the I-Ag7 molecule and the peptide that are essential for docking. This observation indicates that the type 1 diabetes-associated I-Ag7 molecule is structurally malleable and that this plasticity allows the recognition of multiple peptides by individual TCRs that would otherwise be unable to do so.

C-Terminal Extended Hexapeptides as Potent Inhibitors of the NS2B-NS3 Protease of the ZIKA Virus.

The Zika virus (ZIKV) protease is an attractive drug target for the design of novel inhibitors to control the ZIKV infection. As the protease substrate-binding site contains acidic residues, inhibitors with basic residues can be beneficial for the inhibition of protease activities. Molecular dynamics (MD) simulation and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) techniques are employed herein to design potent peptide inhibitors and to understand the nature of the basic residues that can potentially stabilize the acidic residues of the protease substrate-binding site. It is found that the inclusion of K, R, and K at P1, P2, and P3 positions, respectively, and Y at the P4 position (YKRK) would generate a highly stable tetrapeptide-protease complex with a ΔGbind of ~ −80 kcal/mol. We have also shown that the C-terminal extension of this and the second most stable tetrapeptide (YRRR) with small polar residues, such as S and T would generate even more stable hexapeptide-protease complexes. The modes of interactions of these inhibitors are discussed in detail, which are in agreement with earlier experimental studies. Thus, this study is expected to aid in the design of novel antiviral drugs against the ZIKV.

Development of Urea-Bond-Containing Michael Acceptors as Antitrypanosomal Agents Targeting Rhodesain.

Human African Trypanosomiasis (HAT) is a neglected tropical disease widespread in sub-Saharan Africa. Rhodesain, a cysteine protease of Trypanosoma brucei rhodesiense, has been identified as a valid target for the development of anti-HAT agents. Herein, we report a series of urea-bond-containing Michael acceptors, which were demonstrated to be potent rhodesain inhibitors with Ki values ranging from 0.15 to 2.51 nM, and five of them showed comparable k2nd values to that of K11777, a potent antitrypanosomal agent. Moreover, most of the urea derivatives exhibited single-digit micromolar activity against the protozoa, and the presence of substituents at the P3 position appears to be essential for the antitrypanosomal effect. Replacement of Phe with Leu at the P2 site kept unchanged the inhibitory properties. Compound 7 (SPR7) showed the best compromise in terms of rhodesain inhibition, selectivity, and antiparasitic activity, thus representing a new lead compound for future SAR studies.

The distribution of the clinical variables in a population of adult males circumcised for phimosis: A contribution to the clinical classification of phimosis.

The literature regarding the quality of the sex life in adult males after circumcision, due to phimosis, is scarce and sometimes contrasting. This could be due to comparisons of a nonhomogeneous distribution of the clinical variables of men who have undergone circumcision. The objective of this study was to evaluate the distribution of the clinical variables in the adult male population who had circumcision for phimosis, and to propose a clinical classification of the phimosis to characterize it in adult males in more homogeneous sub-groups for the common clinical variables. A population of 244 adult male patients with phimosis was evaluated retrospectively. The mean age was 50.7 years. Each patient was classified according to the most common clinical variables. The variables that make up this classification of the phimosis were: Position (P1-2) to indicate if phimosis is present when the penis is at rest (P2) or only during an erection (P1); Grade (G 0-4) in relation to the extent of glans visibility; Complexity (Co 0-4) of comorbidities; Timespan (T 1-10) of the phimosis. The distribution of the variables was the following: Position P1:30.73%, P2:69.26%; Grade G0:30.73%, G1:23.77%, G2:27.45%, G3:12.29%, G4:5.73%; Complexity (associated penile comorbidities): C0:48.36%, C1:4.5%, C2:0.8%, C3:43.03%, C4:3.27% Timespan: 57.78% of the patients had phimosis for less than a year; 18.03% between 1 and 2 years; 11.88% between 2 and 10 years; and 12.29% for more than 10 years. The distribution of the clinical variables in the adult male population who underwent circumcision due to phimosis was not homogeneous regarding the appearance, severity, comorbidity, and timespan. This non-homogeneity could explain, in some cases, the contrasting results regarding the quality of sex life after circumcision in the literature. The proposed classification can offer an objective tool for researchers and clinicians group the patients into more homogeneous subgroups.

A multi-pronged evaluation of aldehyde-based tripeptidyl main protease inhibitors as SARS-CoV-2 antivirals

As an essential enzyme of SARS-CoV-2, the COVID-19 pathogen, main protease (MPro) is a viable target to develop antivirals for the treatment of COVID-19. By varying chemical compositions at both P2 and P3 positions and the N-terminal protection group, we synthesized 18 tripeptidyl MPro inhibitors that contained also an aldehyde warhead and β-(S-2-oxopyrrolidin-3-yl)-alaninal at the P1 position. Systematic characterizations of these inhibitors were conducted, including their in vitro enzymatic inhibition potency, X-ray crystal structures of their complexes with MPro, their inhibition of MPro transiently expressed in 293T cells, and cellular toxicity and SARS-CoV-2 antiviral potency of selected inhibitors. These inhibitors have a large variation of determined in vitro enzymatic inhibition IC50 values that range from 4.8 to 650 nM. The determined in vitro enzymatic inhibition IC50 values reveal that relatively small side chains at both P2 and P3 positions are favorable for achieving high in vitro MPro inhibition potency, the P3 position is tolerable toward unnatural amino acids with two alkyl substituents on the α-carbon, and the inhibition potency is sensitive toward the N-terminal protection group. X-ray crystal structures of MPro bound with 16 inhibitors were determined. In all structures, the MPro active site cysteine interacts covalently with the aldehyde warhead of the bound inhibitor to form a hemithioacetal that takes an S configuration. For all inhibitors, election density around the N-terminal protection group is weak indicating possible flexible binding of this group to MPro. In MPro, large structural variations were observed on residues N142 and Q189. Unlike their high in vitro enzymatic inhibition potency, most inhibitors showed low potency to inhibit MPro that was transiently expressed in 293T cells. Inhibitors that showed high potency to inhibit MPro transiently expressed in 293T cells all contain O-tert-butyl-threonine at the P3 position. These inhibitors also exhibited relatively low cytotoxicity and high antiviral potency. Overall, our current and previous studies indicate that O-tert-butyl-threonine at the P3 site is a key component to achieve high cellular and antiviral potency for tripeptidyl aldehyde inhibitors of MPro.

Vildagliptin-Derived Dipeptidyl Peptidase 9 (DPP9) Inhibitors: Identification of a DPP8/9-Specific Lead.

Vildagliptin is a marketed DPP4 inhibitor, used in the management of type 2 diabetes. The molecule also has notable DPP8/9 affinity, with some preference for DPP9. Therefore, we aimed to use vildagliptin as a starting point for selective DPP8/9 inhibitors, and to engineer out the parent compound's DPP4-affinity. In addition, we wanted to identify substructures in the obtained molecules that allow their further optimization into inhibitors with maximal DPP9 selectivity. Various 2S-cyanopyrrolidines and isoindoline were investigated as P1 residues of vildagliptin analogs. The obtained set was expanded with derivatives bearing O-substituted, N-(3-hydroxyadamantyl)glycine moieties at the P2 position. In this way, representatives were discovered with DPP8/9 potencies comparable to the parent molecule, but with overall selectivity towards DPP4, DPP2, FAP, and PREP. Furthermore, the most promising molecules in this series have a 4- to 7-fold preference for DPP9 over DPP8. Finally, a molecular dynamics study was carried out to maximize our insight into experimental selectivity data.

A Computational Alanine Scanning of Heptapeptide RRRSAGM Targeting Dengue NS2B/NS3 Protease

The pathogenic dengue virus (DV) transmitted mainly by the Aedes aegypti mosquito has been the most rapidly spreading mosquito-borne viral disease and became a serious threat to global health issue in tropical and sub-tropical countries. The clinical symptoms ranging from mild to a fatal dengue hemorrhagic fever and dengue shock syndrome, characterized by failure of the circulation system that may lead to death. There are still no drugs or vaccines available in the market to prevent or treat dengue infections. Hence, the development of successful drugs that are safe and offer a long-lasting protection against dengue viruses is needed. Recently, the discovery of peptides with high specificity, selectivity, and efficacy; and relatively safer than small-molecule drugs have become a promising agent for a new drug. Previously, a molecular dynamic simulation reported that peptide NRRRRSAGMI from the capsid’s cleavage region had the most hydrogen bonds with NS2B/NS3 protease, thus it leads into a starting linear heptapeptide R(P3)-R(P2)-R(P1)-S(P1’)-A(P2’)-G(P3’)-M(P4’). This study aimed to investigate the importance and specificity of individual amino acid residue of the heptapeptide substrate on the binding affinity and interaction to NS2B/NS3 dengue protease through computational alanine scanning. It showed that the changing of P1 and P2 arginine to alanine resulted to higher values of binding affinity. Thus, the arginine is an important residue at the P1 and P2 positions for the binding of the substrate peptide to DENV NS2B/NS3 protease, hence the amino acid residues which are essential for the enzyme-substrate interactions can be predicted through computational alanine scanning.
 HIGHLIGHTS
 
 Dengue is a mosquito-transmitted virus cause of arthropod-borne viral disease
 The discovery of peptides became a potential agent for dengue virus and previously peptide NRRRRSAGMI has been reported as starting linear heptapeptide RRRSAGM
 A computational alanine scanning based on the peptide RRRSAGM has been performed
 S1 and S2 pockets show strong preferences for basic amino acids, whereas the S3 pocket is less selective for basic residue
 The arginine at P1 and P2 positions of the heptapeptide substrate contribute significantly to the substrate binding affinity
 
 GRAPHICAL ABSTRACT

Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate specificity toward collagen

Collagenase from the gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently even than clostridial collagenase, the most widely used industrial collagenase. However, the structural determinants facilitating this efficiency are unclear. Here, we report the crystal structures of ligand-free and Gly-Pro-hydroxyproline (Hyp)-complexed Ghcol at 2.2 and 2.4 Å resolution, respectively. These structures revealed that the activator and peptidase domains in Ghcol form a saddle-shaped structure with one zinc ion and four calcium ions. In addition, the activator domain comprises two homologous subdomains, whereas zinc-bound water was observed in the ligand-free Ghcol. In the ligand-complexed Ghcol, we found two Gly-Pro-Hyp molecules, each bind at the active site and at two surfaces on the duplicate subdomains of the activator domain facing the active site, and the nucleophilic water is replaced by the carboxyl oxygen of Hyp at the P1 position. Furthermore, all Gly-Pro-Hyp molecules bound to Ghcol have almost the same conformation as Pro-Pro-Gly motif in model collagen (Pro-Pro-Gly)10, suggesting these three sites contribute to the unwinding of the collagen triple helix. A comparison of activities revealed that Ghcol exhibits broader substrate specificity than clostridial collagenase at the P2 and P2′ positions, which may be attributed to the larger space available for substrate binding at the S2 and S2′ sites in Ghcol. Analysis of variants of three active-site Tyr residues revealed that mutation of Tyr564 affected catalysis, whereas mutation of Tyr476 or Tyr555 affected substrate recognition. These results provide insights into the substrate specificity and mechanism of G. hollisae collagenase.

A proteomics-MM/PBSA dual approach for the analysis of SARS-CoV-2 main protease substrate peptide specificity

The main protease Mpro of SARS-CoV-2 is a well-studied major drug target. Additionally, it has been linked to this virus’ pathogenicity, possibly through off-target effects. It is also an interesting diagnostic target. To obtain more data on possible substrates as well as to assess the enzyme’s primary specificity a two-step approach was introduced. First, Terminal Amine Isobaric Labeling of Substrates (TAILS) was employed to identify novel Mpro cleavage sites in a mouse lung proteome library. In a second step, using a structural homology model, the MM/PBSA variant MM/GBSA (Molecular Mechanics Poisson-Boltzmann/Generalized Born Surface Area) free binding energy calculations were carried out to determine relevant interacting amino acids. As a result, 58 unique cleavage sites were detected, including six that displayed glutamine at the P1 position. Furthermore, modeling results indicated that Mpro has a far higher potential promiscuity towards substrates than expected. The combination of proteomics and MM/PBSA modeling analysis can thus be useful for elucidating the specificity of Mpro, and thus open novel perspectives for the development of future peptidomimetic drugs against COVID-19, as well as diagnostic tools.

Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH-DR5-B with Dual VEGFR2 and DR5 Receptor Specificity.

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH–DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH–DR5-B can be considered as a promising candidate for therapeutic applications.

New peptidomimetic rhodesain inhibitors with improved selectivity towards human cathepsins

Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis). Designing selective inhibitors for parasitic cathepsins can be challenging as they share high structural similarities with human cathepsins. In this paper, we describe the development of novel peptidomimetic rhodesain inhibitors by applying a structure-based de novo design approach and molecular docking protocols. The inhibitors with a new scaffold in P2 and P3 position display high selectivity towards trypanosomal rhodesain over human cathepsins L and B and high antitrypanosomal activity. Vinylsulfonate 2a has emerged as a potent rhodesain inhibitor (k2nd = 883 • 103 M−1 s−1) with single-digit nanomolar binding affinity (Ki = 9 nM) and more than 150-fold selectivity towards human cathepsins and it thus constitutes an interesting starting compound for the further development of selective drugs against Human African Trypanosomiasis.

Stress–Strain Analysis of S-Shaped Welded Metal Bellows Under Axial Load Based on Finite Element Analysis and Experimental Validation

Welded metal bellows are commonly used in mechanical sealing devices. Its properties directly affect the sealing effect, and thus it is very important to study its stress–strain for a high reliability. In this study, to explore the stress–strain state of the diaphragm of welded metal bellows under axial force, based on the elastic mechanics, S-shaped welded bellows of single-layer (SL) and double-layer (DL) with small diameters (D1, size 60 mm) and large diameter (D2, size 95 mm) were used as materials. ANSYS Workbench was used for simulation, and a digital image correlation (DIC) method was carried out for experimental validation. The results of finite element analysis (FEA) showed that the greater the compression, the greater the strain. The DIC analysis showed that the strain increased with the increase of the amount of compression. However, the diaphragm of DL metal bellows showed a lower stress than that of the SL metal bellows. Moreover, the strain of S-shaped welded bellows at larger-diameter was less than at small-diameter. This trend applied to single and double layers. In some layers, the strain showed abrupt changes. The Principal strain was 0.02563 at P6 position of SL-D1 and 0.01333 at P8 position of DL-D1 with compression at 5 mm; while with compression at 4 mm, the Principal strain was 0.0841 at P8 position of SL-D2 and 0.00182 at P6 position of DL-D2. In conclusion, the resistance to elastic deformation of double-layer S-shaped welded metal bellows is better than that of single-layer. Moreover, this study provides a new idea and method for the strain test of S-shaped welded metal bellows.

Implementation of a Four-Class Motor Imagery Brain Computer Interface Using Long Short-Term Memory Neural Network

We have developed multi-channel dry-electrode electroencephalography (EEG) system to implement a brain computer interface (BCI) and discriminate four-class motor imagery signals. The EEG channels recorded from Fz, F3, F4, C3, Cz, C4, P3, and P4 positions, according to the international 10‒20 EEG system, were acquired and wirelessly transmitted to a personal computer. Five subjects were recruited to perform right hand, left hand, right foot, and left foot imagery movements with 60 trials in each movement type. EEG data were segmented into epochs from 0 to 8 s and anchored to time points of imagery movement cues. The segmented EEG epochs were pre-processed using the Morlet wavelet. A long short-term memory (LSTM) neural network with 64 LSTM cells was constructed to discriminate EEG signals recorded from different imagery movements. Among total EEG epochs, 80% were randomly chosen as training data and the rest 20% were used as validation data. The detection accuracy reached 89%.

A new blind genus of Anurophorinae (Collembola: Isotomidae) with seven anal spines from Northwest China.

A new genus and species of Anurophorinae, Heptacanthella yinae gen. et sp. nov., is described from Xinjiang Province, Northwest China. The new genus has four spines on Abd. V in p1 and p2 position and three spines on Abd. VI in m0 and m1 position, seven anal spines in all distally on the abdomen. It can be distinguished from other spined genera of the subfamily by the position and number of the anal spines. A comparison of spinal armament of last abdominal segments in 13 genera of Anurophorinae is made here. Heptacanthella yinae gen. et sp. nov. is also characterised by the absence of eyes, pigment, furca and retinaculum, presence of reticulated cuticle and an s-formula of 33/22224 (s) and 10/001 (ms). The value of anal spines in taxonomy of Isotomidae is discussed.

Disposition of Nirmatrelvir, an Orally Bioavailable Inhibitor of SARS-CoV-2 3C-Like Protease, across Animals and Humans.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), was recently granted emergency use authorization by multiple regulatory agencies for the treatment of coronavirus disease 2019 (COVID-19) in adults and pediatric patients. Disposition studies on nirmatrelvir in animals and in human reagents, which were used to support clinical studies, are described herein. Plasma clearance was moderate in rats (27.2 ml/min per kg) and monkeys (17.1 ml/min per kg), resulting in half-lives of 5.1 and 0.8 hours, respectively. The corresponding oral bioavailability was moderate in rats (34%-50%) and low in monkeys (8.5%), primarily due to oxidative metabolism along the gastrointestinal tract in this species. Nirmatrelvir demonstrated moderate plasma protein binding in rats, monkeys, and humans with mean unbound fractions ranging from 0.310 to 0.478. The metabolism of nirmatrelvir was qualitatively similar in liver microsomes and hepatocytes from rats, monkeys, and humans; prominent metabolites arose via cytochrome P450 (CYP450)-mediated oxidations on the P1 pyrrolidinone ring, P2 6,6-dimethyl-3-azabicyclo[3.1.0]hexane, and the tertiary-butyl group at the P3 position. Reaction phenotyping studies in human liver microsomes revealed that CYP3A4 was primarily responsible (fraction metabolized = 0.99) for the oxidative metabolism of nirmatrelvir. Minor clearance mechanisms involving renal and biliary excretion of unchanged nirmatrelvir were also noted in animals and in sandwich-cultured human hepatocytes. Nirmatrelvir was a reversible and time-dependent inhibitor as well as inducer of CYP3A activity in vitro. First-in-human pharmacokinetic studies have demonstrated a considerable boost in the oral systemic exposure of nirmatrelvir upon coadministration with the CYP3A4 inhibitor ritonavir, consistent with the predominant role of CYP3A4 in nirmatrelvir metabolism. SIGNIFICANCE STATEMENT: The manuscript describes the preclinical disposition, metabolism, and drug-drug interaction potential of PF-07321332 (nirmatrelvir), an orally active peptidomimetic-based inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease, which has been granted emergency use authorization by multiple regulatory agencies around the globe for the treatment of coronavirus disease 2019 (COVID-19) in COVID-19-positive adults and pediatric patients who are at high risk for progression to severe COVID-19, including hospitalization or death.

SAR evolution towards potent C-terminal carboxamide peptide inhibitors of Zika virus NS2B-NS3 protease

Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC50 = 30 nM) with high selectivity against trypsin-like proteases and the proteases of other flavivirus, such as Dengue 2 virus (DEN2V) and West Nile virus (WNV).