Deciphering complex mechanisms of resistance and loss of potency through coupled molecular dynamics and machine learning [preprint]

Drug resistance threatens many critical therapeutics through mutations in the drug target. The molecular mechanisms by which combinations of mutations, especially involving those distal from the active site, alter drug binding to confer resistance are poorly understood and thus difficult to counteract. A strategy coupling parallel molecular dynamics simulations and machine learning to identify conserved mechanisms underlying resistance was developed. A series of 28 HIV-1 protease variants with up to 24 varied substitutions were used as a rigorous model of this strategy. Many of the mutations were distal from the active site and the potency to darunavir varied from low pM to near μM. With features extracted from molecular dynamics simulations, elastic network machine learning was applied to correlate physical interactions at the molecular level with potency loss. This fit to within 1 kcal/mol of experimental potency for both the training and test sets, outperforming MM/GBSA calculations. Feature reduction resulted in a model with 4 specific features that correspond to interactions critical for potency regardless of enzyme variant. These predictive features throughout the enzyme would not have been identified without dynamics and machine learning and specifically varied with potency. This approach enables capturing the conserved dynamic molecular mechanisms by which complex combinations of mutations confer resistance and identifying critical interactions which serve as bellwethers over a wide range of inhibitor potency. Machine learning models leveraging molecular dynamics can thus elucidate mechanisms that confer loss of affinity due to variations distal from the active site, such as in drug resistance

HIV-1 protease-substrate coevolution in nelfinavir resistance

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30\u27s interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. IMPORTANCE: Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations

Serum IL-1β, IL-6, IL-8, and TNF-α Levels in Early Diagnosis and Management of Neonatal Sepsis

Aim. To determine serum IL-1β, IL-6, IL-8, and TNF-α levels in neonatal sepsis at the time of diagnosis and after therapy, and to show the meaningful on the follow up. Methods. This prospective study was performed on newborns who were hospitalized for neonatal sepsis and who were classified as culture-proven sepsis (n=12), as culture-negative sepsis (n=21), and as healthy newborns (n=17). Results. At the time of diagnosis, serum IL-1β, IL-6, IL-8, and TNF-α levels of culture-proven sepsis were significantly higher than those of the control groups (P<.05). At the time of diagnosis, IL-1β, IL-6, IL-8, and TNF-α levels of culture-proven sepsis and culture-negative sepsis were significantly higher than levels at the seventh day after antibiotic treatment. Conclusion. Serum IL-1β, IL-6, IL-8, and TNF-α are mediators of inflammation and can be used at the diagnosis and at the evaluation of the therapeutic efficiency in neonatal sepsis

Interdependence of Inhibitor Recognition in HIV-1 Protease

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1\u27 and S2\u27 subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1\u27 subsite highly influences other subsites: the extension of the hydrophobic P1\u27 moiety results in 1) reduced van der Waals contacts in the P2\u27 subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor

The Levels of Ghrelin, TNF-α, and IL-6 in Children with Cyanotic and Acyanotic Congenital Heart Disease

Background/Aim. Ghrelin has effects on nutrient intake and growth. The cause of growth retardation in congenital heart disease is multifactorial. The aim of the present study is to investigate the ghrelin in congenital heart disease and the association of ghrelin with TNF-α and IL-6. Materials and methods. We measured serum ghrelin, TNF-α, and IL-6 levels using spesific immunoassay in 68 patients (47 acyanotic, 21 cyanotic with congenital heart disease) and in 25 control subjects. Results. In comparison to controls, serum ghrelin, TNF-α levels were significantly higher in acyanotic patients and cyanotic patients with congenital heart disease (P<.0001). In acyanotic and cyanotic patients with congenital heart disease, there was a positive correlation between ghrelin and TNF-α (r=.485, P<.05 and r=.573, P<.01, resp.). Conclusion. Serum ghrelin levels is elevated in acyanotic and cyanotic patients with congenital heart disease. Increased ghrelin levels represents malnutrition and growth retardation in these patients. The relation of ghrelin with cytokines may be explained by the possible effect of chronic congestive heart failure and chronic shunt hypoxemia

Serum Endothelin-1 and Transforming Growth Factor-β Levels in the Newborns With Respiratory Distress

The purpose of this present study was to evaluate the serum levels of ET-1 and TGF-β in the newborns with respiratory distress. In this study, newborns with respiratory distress hospitalized into the Newborn Intensive Care Unit were included. The highest values of ET-1 and TGF-β were obtained from newborns with diagnosis as meconium aspiration syndrome (5.70 ± 5.87 pg/mL and 3.75 ± 1.94 pg/mL, resp) in the sample obtained in the first six hours after birth, and these are statistically different from control group (P < .05). Also, same results were obtained for newborns with respiratory distress syndrome (3.37 ± 1.59 pg/mL and 2.05 ± 0.98 pg/mL, resp). After oxygen treatment, ET-1 values obtained in the first six hours of life were decreased regularly in the following days (P < .05). In the differentiating diagnosis of the respiratory distress of newborns, the investigation of ET-1 and TGF-β levels is meaningful. The ET-1 levels investigated in the first six hours is more useful in determining the prognosis, and repeating ET-1 levels in the following days is more meaningful to determine clinical response

Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s

The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin\u27s high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 muM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE: Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti, continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors

Mechanism for APOBEC3G catalytic exclusion of RNA and non-substrate DNA

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2\u27-deoxy-2\u27-fluorine substituted cytidines, we show that a 2\u27-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2\u27-hydroxyl group destabilizes the pi-pi stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2\u27-hydroxyl group

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A\u27s involvement in mutation of endogenous or exogenous DNA