Biology subjects

Wlodawer, A.

Publications and source records attributed to Wlodawer, A..

5 recordsLinked to original sources

bioRxiv2021

Molecular insights into the inhibition of plasmepsins by HIV-1 protease inhibitors: Implications for antimalarial drug discovery

1.Malaria is a deadly disease, and the worldwide emergence of drug resistance in the Plasmodium parasites demands the development of novel and potent antimalarials. HIV-1 protease inhibitors (HIV-1 PIs) alleviate the Plasmodium pathogenesis during malaria/HIV-1 co-infection plausibly by inhibiting vacuolar plasmepsins (PMs), the hemoglobin degrading proteases from P. falciparum. All five FDA-approved HIV-1 PIs tested against PMII exhibit the Ki values in the low micromolar range of which RTV and LPV display the highest inhibition activity. Both inhibitors impede in vitro growth of P. falciparum at low micromolar IC50 values. We report the first crystal structures of PMII complexed with RTV and LPV that reveal the binding mode and interactions of the inhibitors in the active site as well as elucidate the mechanism underlying their differential potency. The conformational flexibility of the P4 group in RTV allows it to explore multiple regions of the S4 pocket. The present study establishes vacuolar PMs as potential drug targets of HIV-1 PIs. The molecular details explaining the inhibitory mechanism of HIV-1 PIs might be crucial in designing novel and potent antimalarial analogs. This work strengthens the prospect of drug repurposing as an alternative strategy towards antimalarial treatments and provides an opportunity to tackle malaria and HIV-1 co-infection.

biochemistry↗

bioRxiv2021

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme are involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

biophysics↗

bioRxiv2021

Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Proteinco-immunization in rhesus macaques

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The DNA-only vaccine regimens were compared to a regimen that included co- immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques. Author summaryAnti-Spike neutralizing antibodies provide strong protection against SARS-CoV-2 infection in animal models, and correlate with protection in humans, supporting the notion that induction of strong humoral immunity is key to protection. We show induction of robust antibody and T cell responses by different Spike DNA-based vaccine regimens able to effectively mediate protection and to control SARS-CoV-2 infection in the rhesus macaque model. This study provides the opportunity to compare vaccines able to induce different humoral and cellular immune responses in an effort to develop durable immunity against the SARS-CoV-2. A vaccine regimen comprising simultaneous co-immunization of DNA and Protein at the same anatomical site showed best neutralizing abilities and was more effective than DNA alone in inducing protective immune responses and controlling SARS-CoV-2 infection. Thus, an expansion of the DNA vaccine regimen to include co-immunization with Spike protein may be of advantage also for SARS-CoV-2.

immunology↗

bioRxiv2020

Convergent evolution of the Hedgehog/Intein fold in protein splicing

The widely used molecular evolutionary clock assumes the divergent evolution of proteins. Convergent evolution has been proposed only for small protein elements but not for an entire protein fold. We investigated the structural basis of the protein splicing mechanism by class 3 inteins, which is distinct from class 1 and 2 inteins. We gathered structural and mechanistic evidence supporting the notion that the Hedgehog/INTein (HINT) superfamily fold, commonly found in protein splicing and related phenomena, could be an example of convergent evolution of an entire protein fold. We propose that the HINT fold is a structural and biochemical solution for trans-peptidyl and trans-esterification reactions.

biochemistry↗

bioRxiv2019

The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from a cis-splicing intein

Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. The 1.0 [A]-resolution crystal structure of a variant from naturally split gp41-1 intein, identified from the environmental metagenomic sequence data, revealed an improved pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N-and C-terminal intein fragments. We successfully created orthogonal split inteins by engineering a similar charge network in the same region of a cis-splicing intein. The same strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins.

biochemistry↗