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It is established that reverse hydroxamate analogs of fosmidomycin inhibit the growth of Plasmodium falciparum by inhibiting 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), the second enzyme of the non-mevalonate pathway, which is absent in humans. Recent biochemical studies have demonstrated that novel reverse fosmidomycin analogs with phenylalkyl substituents at the hydroxamate nitrogen exhibit inhibitory activities against PfDXR at the nanomolar level. Moreover, crystallographic analyses have revealed that the phenyl moiety of the N-phenylpropyl substituent is accommodated in a previously unidentified subpocket within the active site of PfDXR. In this study, the crystal structures of PfDXR in complex with a series of reverse N-phenylalkyl derivatives of fosmidomycin were determined to ascertain whether the high inhibitory activities of the derivatives are consistently attributable to the utilization of the subpocket of PfDXR. While all reverse fosmidomycin derivatives with an N-substituted phenylalkyl group exhibit potent inhibitory activity against PfDXR, the present crystal structure analyses revealed that their binding modes to the PfDXR are not uniform. In these compounds, the nanomolar inhibitory activities appear to be driven by binding modes distinct from that observed for the inhibitor containing the N-phenylpropyl group. The structural information obtained in this study will provide a basis for further design of fosmidomycin derivatives. [ABSTRACT FROM AUTHOR]

Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1′-P2′...]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Å resolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84 Å, α = 67.42, β = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry ) as a template and refinement of the structure is in progress. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) preferentially cleaves substrate peptides with Asp and Glu at the P1 position [NH2-P2-P1(Asp/Glu)-P1′-P2′...]. For crystallographic studies, PgDPP11 was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.82 Å resolution were collected from an orthorhombic crystal form belonging to space group C2221, with unit-cell parameters a = 99.33, b = 103.60, c = 177.33 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress. [ABSTRACT FROM AUTHOR]

Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P212121, with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress. [ABSTRACT FROM AUTHOR]

Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-β- d-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of ∼105 kDa (Gls93-F1) and ∼100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 Å, and diffracted to 1.8 Å resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P212121, with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 Å, and diffracted to 2.4 Å resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution. [ABSTRACT FROM AUTHOR]

Previously, we purified a 59-kDa protein that binds to the κB motif of the Sarcophaga lectin gene. Here we report its cDNA cloning and some of its characteristics as a novel member of the Rel/Ankyrin-family. This protein, named SRAM, contained a Rel homology domain, a nuclear localization signal and 4 ankyrin repeats, but lacked the Ser-rich domain and PEST sequence that Relish contained. We found that SRAM was localized in the nuclei of NIH-Sape-4 cells, which are an embryonic cell line of Sarcophaga. The Sarcophaga lectin gene promoter containing tandem repeats of the κB motifs was activated in NIH-Sape-4 cells. In Drosophila mbn-2 cells, Dif alone activated this reporter gene and a cooperative effect was detected when SHAM and Dif were co-transfected, although SEAM alone did not activate it. This is the first report of a Rel/Ankyrin molecule that exists in the nuclei. [ABSTRACT FROM AUTHOR]

Multidrug-resistant pathogens pose a major threat to human health, necessitating the identification of new drug targets and lead compounds that are not susceptible to cross-resistance. This study demonstrates that novel reverse thia analogs of the phosphonohydroxamic acid antibiotic fosmidomycin inhibit 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme for Plasmodium falciparum , Escherichia coli , and Mycobacterium tuberculosis that is absent in humans. Some novel analogs with large α-phenyl substituents exhibited strong inhibition across these three DXR orthologues, surpassing the inhibitory activity of fosmidomycin. Despite nanomolar target inhibition, the new DXR inhibitors demonstrated mainly weak or no in vitro growth inhibition of the pathogens. Crystallographic studies revealed that compounds 12a and 12b induce an open Pf DXR conformation and that the enzyme selectively binds the S -enantiomers. The study underscores the difficulties of achieving potent cellular activity despite strong DXR inhibition and emphasizes the need for novel structural optimization strategies and comprehensive pharmacokinetic studies., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum . Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar Pf DXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N -phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N -terminal domain. As shown for reverse carba and thia analogs, Pf DXR selectively binds the S -enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

Aim: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR)., Methods: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs., Results: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M., Conclusion: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is found mainly in the central nervous system of vertebrates and catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides in vitro. Recently, CNP has been identified as a member of the 2H phosphoesterase superfamily. Here we have determined the crystal structure of the catalytic fragment of human CNP (hCNP-CF) at 1.3 A resolution.