Biology subjects

Yilmaz, N. K.

Publications and source records attributed to Yilmaz, N. K..

4 recordsLinked to original sources

bioRxiv2024

Direct delivery of stabilized Cas-embedded base editors achieves efficient and accurate editing of clinically relevant targets

Over the last 5 years, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, in part due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study we engineeredhighly stabilized Cas-embedded CBEs (sCE_CBEs) that integrate several recent advances, andthat are highly expressible and soluble for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting sCE_CBE RNP complexes efficiently and specifically target TC dinucleotides with minimal off-target or bystander mutations. Additional uracil glycosylase inhibitor (UGI) protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation to untreated levels. A single electroporation was sufficient to effectively edit the therapeutically relevant locus for sickle cell disease in hematopoietic stem and progenitor cells (HSPC) in a dose dependent manner without cellular toxicity. Significantly, these sCE_CBE RNPs permitted for the transplantation of edited HSPCs confirming highly efficient editing in engrafting hematopoietic stem cells in mice. The success of the designed sCBE editors, with improved solubility and enhanced on-target editing, demonstrates promising agents for cytosine base editing at other disease-related sites in HSPCs and other cell types.

biochemistry↗

bioRxiv2022

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.

molecular biology↗

bioRxiv2021

Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

biochemistry↗

bioRxiv2019

Selection of HIV-1 for Resistance to Fourth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in the high drug concentrations that are achived in vivo. Little is known about the de novo resistance pathway for DRV. We have selected for resistance to high drug concentrations against ten PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small chemical changes in the inhibitor P1 side chain led to preferential use of one pathway over the other. Changes in the inhbitor P2 side chain determined differences in potency that were retained in the resistant viruses. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fourth generation PIs, and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.

microbiology↗