An alternative strategy for inhibiting multidrug-resistant mutants of the dimeric HIV-1 protease by targeting the subunit interface

2007 ◽  
Vol 35 (3) ◽  
pp. 551-554 ◽  
Author(s):  
L. Bannwarth ◽  
M. Reboud-Ravaux
Keyword(s):  
Small Molecules ◽  
Protein Interactions ◽  
Active Site ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Mechanism Of Inhibition ◽  
Therapeutic Molecules ◽  
Β Sheet ◽  
Hiv 1

Mutations that occur in response to the HIV-1 protease inhibitors are responsible for the development of multidrug cross-resistance to these antiproteases in AIDS treatment. One alternative to inhibiting the active site of HIV-1 protease is to target the dimer interface of the homodimeric enzyme at the antiparallel β-sheet formed by the interdigitation of the C- and N-ends of each monomer. This region is highly conserved and is responsible for approx. 75% of the dimer-stabilization energy. The strategies that have been used to design small molecules to target the interface antiparallel β-sheet have produced lipopeptides, guanidinium derivatives and peptides (or peptidomimetics) cross-linked with spacers. The mechanism of inhibition was determined using a combination of kinetic and biophysical methods. These dimerization inhibitors proved equally active in vitro against both wild-type and mutated proteases. They are therefore promising alternatives to active-site-directed inhibitors in AIDS therapy. Disruption of protein–protein interactions by small molecules is a new way to obtain potentially therapeutic molecules.

scholarly journals 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1997 ◽  
Vol 41 (5) ◽  
pp. 1082-1093 ◽  
Author(s):  
S M Daluge ◽  
S S Good ◽  
M B Faletto ◽  
W H Miller ◽  
M H St Clair ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Oral Bioavailability ◽  
Human Peripheral Blood ◽  
Cross Resistance ◽  
Immunodeficiency Virus ◽  
Carbocyclic Nucleoside ◽  
Hiv 1 ◽  
In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals In Vitro Activity of the Novel Pleuromutilin Lefamulin (BC-3781) and Effect of Efflux Pump Inactivation on Multidrug-Resistant and Extensively Drug-Resistant Neisseria gonorrhoeae

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Susanne Jacobsson ◽  
Susanne Paukner ◽  
Daniel Golparian ◽  
Jörgen S. Jensen ◽  
Magnus Unemo
Keyword(s):  
Efflux Pump ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Drug Resistant ◽  
The Novel ◽  
Extensively Drug Resistant ◽  
Optimal Dosing ◽  
And Performance ◽  
Reference Strains

ABSTRACT We evaluated the activity of the novel semisynthetic pleuromutilin lefamulin, inhibiting protein synthesis and growth, and the effect of efflux pump inactivation on clinical gonococcal isolates and reference strains (n = 251), including numerous multidrug-resistant and extensively drug-resistant isolates. Lefamulin showed potent activity against all gonococcal isolates, and no significant cross-resistance to other antimicrobials was identified. Further studies of lefamulin are warranted, including in vitro selection and mechanisms of resistance, pharmacokinetics/pharmacodynamics, optimal dosing, and performance in randomized controlled trials.

scholarly journals In Vitro Antiviral Activity and Cross-Resistance Profile of PL-100, a Novel Protease Inhibitor of Human Immunodeficiency Virus Type 1

2007 ◽  
Vol 51 (11) ◽  
pp. 4036-4043 ◽  
Author(s):  
Serge Dandache ◽  
Guy Sévigny ◽  
Jocelyn Yelle ◽  
Brent R. Stranix ◽  
Neil Parkin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Highly Active Antiretroviral Therapy ◽  
Cross Resistance ◽  
Drug Resistant ◽  
Resistance Profile ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Despite the success of highly active antiretroviral therapy, the current emergence and spread of drug-resistant variants of human immunodeficiency virus (HIV) stress the need for new inhibitors with distinct properties. We designed, produced, and screened a library of compounds based on an original l-lysine scaffold for their potentials as HIV type 1 (HIV-1) protease inhibitors (PI). One candidate compound, PL-100, emerged as a specific and noncytotoxic PI that exhibited potent inhibition of HIV-1 protease and viral replication in vitro (Ki , ∼36 pM, and 50% effective concentration [EC50], ∼16 nM, respectively). To confirm that PL-100 possessed a favorable resistance profile, we performed a cross-resistance study using a panel of 63 viral strains from PI-experienced patients selected for the presence of primary PI mutations known to confer resistance to multiple PIs now in clinical use. The results showed that PL-100 retained excellent antiviral activity against almost all of these PI-resistant viruses and that its performance in this regard was superior to those of atazanavir, amprenavir, indinavir, lopinavir, nelfinavir, and saquinavir. In almost every case, the increase in the EC50 for PL-100 observed with viruses containing multiple mutations in protease was far less than that obtained with the other drugs tested. These data underscore the potential for PL-100 to be used in the treatment of drug-resistant HIV disease and argue for its further development.

scholarly journals Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

2018 ◽  
Vol 74 (12) ◽  
pp. 765-769 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Chi-Hung Huang ◽  
Shu-Jung Lai ◽  
Yeh Chen
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Multidrug Resistant ◽  
Structural Basis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Peptidoglycan Synthesis ◽  
C Terminus ◽  
Dimeric Protein ◽  
Β Sheet

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

scholarly journals Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

2012 ◽  
Vol 78 (9) ◽  
pp. 3087-3097 ◽  
Author(s):  
Orla Condell ◽  
Carol Iversen ◽  
Shane Cooney ◽  
Karen A. Power ◽  
Ciara Walsh ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Food Industry ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Antimicrobial Compounds ◽  
Cross Tolerance ◽  
Content Type ◽  
Food Grade ◽  
High Level

ABSTRACTBiocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR)Salmonella entericastrains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds ofin vitroselection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure ofSalmonellastrains to an active biocidal compound, a high-level of tolerance was selected for a number ofSalmonellaserotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonicSalmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.

scholarly journals Lead Optimization of Dehydroemetine for Repositioned Use in Malaria

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Priyanka Panwar ◽  
Kepa K. Burusco ◽  
Muna Abubaker ◽  
Holly Matthews ◽  
Andrey Gutnov ◽  
...  
Keyword(s):  
De Novo ◽  
Antimalarial Activity ◽  
Drug Repositioning ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Synthetic Analogue ◽  
80S Ribosome ◽  
Content Type ◽  
Resistant Strains

ABSTRACT Drug repositioning offers an effective alternative to de novo drug design to tackle the urgent need for novel antimalarial treatments. The antiamoebic compound emetine dihydrochloride has been identified as a potent in vitro inhibitor of the multidrug-resistant strain K1 of Plasmodium falciparum (50% inhibitory concentration [IC50], 47 nM ± 2.1 nM [mean ± standard deviation]). Dehydroemetine, a synthetic analogue of emetine dihydrochloride, has been reported to have less-cardiotoxic effects than emetine. The structures of two diastereomers of dehydroemetine were modeled on the published emetine binding site on the cryo-electron microscopy (cryo-EM) structure with PDB code 3J7A (P. falciparum 80S ribosome in complex with emetine), and it was found that (−)-R,S-dehydroemetine mimicked the bound pose of emetine more closely than did (−)-S,S-dehydroisoemetine. (−)-R,S-dehydroemetine (IC50 71.03 ± 6.1 nM) was also found to be highly potent against the multidrug-resistant K1 strain of P. falciparum compared with (−)-S,S-dehydroisoemetine (IC50, 2.07 ± 0.26 μM), which loses its potency due to the change of configuration at C-1′. In addition to its effect on the asexual erythrocytic stages of P. falciparum, the compound exhibited gametocidal properties with no cross-resistance against any of the multidrug-resistant strains tested. Drug interaction studies showed (−)-R,S-dehydroemetine to have synergistic antimalarial activity with atovaquone and proguanil. Emetine dihydrochloride and (−)-R,S-dehydroemetine failed to show any inhibition of the hERG potassium channel and displayed activity affecting the mitochondrial membrane potential, indicating a possible multimodal mechanism of action.

scholarly journals Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
James O’Connell ◽  
John Porter ◽  
Boris Kroeplien ◽  
Tim Norman ◽  
Stephen Rapecki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Protein Interactions ◽  
General Mechanism ◽  
Protein Protein Interactions ◽  
Biologic Drugs ◽  
Small Molecule Drugs ◽  
Necrosis Factor

AbstractTumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn’s disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein–protein interactions.

scholarly journals Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

2004 ◽  
Vol 78 (6) ◽  
pp. 3123-3132 ◽  
Author(s):  
Bradley C. Logsdon ◽  
John F. Vickrey ◽  
Philip Martin ◽  
Gheorghe Proteasa ◽  
Jay I. Koepke ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Surface Plasmon Resonance ◽  
Protease Inhibitors ◽  
Surface Plasmon ◽  
Active Site ◽  
Multidrug Resistant ◽  
Immunodeficiency Virus ◽  
Active Site Cavity ◽  
Hiv 1

ABSTRACT The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-angstrom (Å) crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease “flaps” stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 Å. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1′, S3, and S3′ pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k off rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k on and k off data (Kd = k off/k on) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

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