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To date, there are no specific treatment regimens for HIV-1-related central nervous system (CNS) complications, such as HIV-1-associated neurocognitive disorders (HAND). Here, we report that two newly generated CNS-targeting HIV-1 protease (PR) inhibitors (PIs), GRL-08513 and GRL-08613, which have a P1-3,5-bis-fluorophenyl or P1-para-monofluorophenyl ring and P2-tetrahydropyrano-tetrahydrofuran (Tp-THF) with a sulfonamide isostere, are potent against wild-type HIV-1 strains and multiple clinically isolated HIV-1 strains (50% effective concentration [EC(50)]: 0.0001 to ∼0.0032 μM). As assessed with HIV-1 variants that had been selected in vitro to propagate at a 5 μM concentration of each HIV-1 PI (atazanavir, lopinavir, or amprenavir), GRL-08513 and GRL-08613 efficiently inhibited the replication of these highly PI-resistant variants (EC(50): 0.003 to ∼0.006 μM). GRL-08513 and GRL-08613 also maintained their antiviral activities against HIV-2(ROD) as well as severely multidrug-resistant clinical HIV-1 variants. Additionally, when we assessed with the in vitro blood-brain barrier (BBB) reconstruction system, GRL-08513 and GRL-08613 showed the most promising properties of CNS penetration among the evaluated compounds, including the majority of FDA-approved combination antiretroviral therapy (cART) drugs. In the crystallographic analysis of compound-PR complexes, it was demonstrated that the Tp-THF rings at the P2 moiety of GRL-08513 and GRL-08613 form robust hydrogen bond interactions with the active site of HIV-1 PR. Furthermore, both the P1-3,5-bis-fluorophenyl- and P1-para-monofluorophenyl rings sustain greater contact surfaces and form stronger van der Waals interactions with PR than is the case with darunavir-PR complex. Taken together, these results strongly suggest that GRL-08513 and GRL-08613 have favorable features for patients infected with wild-type/multidrug-resistant HIV-1 strains and might serve as candidates for a preventive and/or therapeutic agent for HAND and other CNS complications.

Coronavirus disease-2019 (COVID-19) is a pandemic with a high morbidity rate occurring over recent years. COVID-19 is caused by the severe acute respiratory syndrome causing coronavirus type-2 (SARS-CoV-2). COVID-19 not only challenged mankind but also gave scope to the evolution of various vaccine design technologies. Although these vaccines protected and saved many lives, with the emerging viral strains, some of the strains may pose a threat to the currently existing vaccine design that is primarily based on the wild type spike protein of SARS-CoV-2. To evaluate the risk involved from such mutant viral strains, we performed a systematic in silico amino acid substitution of critical residues in the receptor binding domain (RBD) of the spike protein. Our molecular modeling analysis revealed significant topological changes in the RBD of spike protein suggesting that they could potentially contribute to the loss of antigen specificity for the currently existing therapeutic antibodies/vaccines, thus posing a challenge to the current vaccine strategies that are based on wild type viral spike protein epitopes. The structural deviations discussed in this article should be considered carefully in the future vaccine design.

We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2′-methoxybenzene, and a sulfonamide isostere, is highly active against laboratory and primary clinical HIV-1 isolates (EC50: 0.0014–0.0028 μM) with minimal cytotoxicity (CC50: 39.0 μM). Similarly, GRL-09510 efficiently blocked the replication of HIV-1NL4-3 variants, which were capable of propagating at high-concentrations of atazanavir, lopinavir, and amprenavir (APV). GRL-09510 was also potent against multi-drug-resistant clinical HIV-1 variants and HIV-2ROD. Under the selection condition, where HIV-1NL4-3 rapidly acquired significant resistance to APV, an integrase inhibitor raltegravir, and a GRL-09510 congener (GRL-09610), no variants highly resistant against GRL-09510 emerged over long-term in vitro passage of the virus. Crystallographic analysis demonstrated that the Crwn-THF moiety of GRL-09510 forms strong hydrogen-bond-interactions with HIV-1 protease (PR) active-site amino acids and is bulkier with a larger contact surface, making greater van der Waals contacts with PR than the bis-THF moiety of darunavir. The present data demonstrate that GRL-09510 has favorable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants, that the newly generated P2-Crwn-THF moiety confers highly desirable anti-HIV-1 potency. The use of the novel Crwn-THF moiety sheds lights in the design of novel PIs.

There is currently no specific therapeutics for the HIV-1-related central nervous system (CNS) complications. Here we report that three newly designed CNS-targeting HIV-1 protease inhibitors (PIs), GRL-083-13, GRL-084-13, and GRL-087-13, which contain a P1-3,5-bis-fluorophenyl or P1-para-monofluorophenyl ring, and P2-bis-tetrahydrofuran (bis-THF) or P2-tetrahydropyrano-tetrahydrofuran (Tp-THF), with a sulfonamide isostere, are highly active against wild-type HIV-1 strains and primary clinical isolates (50% effective concentration [EC(50)], 0.0002 to ∼0.003 μM), with minimal cytotoxicity. These CNS-targeting PIs efficiently suppressed the replication of HIV-1 variants (EC(50), 0.002 to ∼0.047 μM) that had been selected to propagate at high concentrations of conventional HIV-1 PIs. Such CNS-targeting PIs maintained their antiviral activity against HIV-2(ROD) as well as multidrug-resistant clinical HIV-1 variants isolated from AIDS patients who no longer responded to existing antiviral regimens after long-term therapy. Long-term drug selection experiments revealed that the emergence of resistant-HIV-1 against these CNS-targeting PIs was substantially delayed. In addition, the CNS-targeting PIs showed the most favorable CNS penetration properties among the tested compounds, including various FDA-approved anti-HIV-1 drugs, as assessed with the in vitro blood-brain barrier reconstruction system. Crystallographic analysis demonstrated that the bicyclic rings at the P2 moiety of the CNS-targeting PIs form strong hydrogen-bond interactions with HIV-1 protease (PR) active site. Moreover, both the P1-3,5-bis-fluorophenyl and P1-para-monofluorophenyl rings sustain greater van der Waals contacts with PR than in the case of darunavir (DRV). The data suggest that the present CNS-targeting PIs have desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 strains and might serve as promising preventive and/or therapeutic candidates for HIV-1-associated neurocognitive disorders (HAND) and other CNS complications.

We report here that GRL-10413, a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a hydroxyethylamine sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC 50 ] of 0.00035 to 0.0018 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC 50 ] = 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1 NL4-3 variants selected by use of atazanavir, lopinavir, or amprenavir (APV) at concentrations of up to 5 μM (EC 50 = 0.0021 to 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that against APV. In addition, GRL-10413 showed favorable central nervous system (CNS) penetration properties as assessed with an in vitro blood-brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants, with favorable CNS penetration capability, and that the newly modified P1 moiety may confer desirable features in designing novel anti-HIV-1 PIs.

We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1 WT ), with 50% effective concentrations (EC 50 s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC 50 ) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIV MDR ). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1 DRV R P51 ); the three compounds remained active against HIV-1 DRV R P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIV MDR . IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIV DRV R ) has recently been observed in vivo and in vitro . Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIV DRV R . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIV DRV R . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIV DRV R .

Photoreceptors of the squid Loligo pealei contain a G-protein-coupled receptor (GPCR) signaling system that activates phospholipase C in response to light. Analogous to the mammalian visual system, signaling of the photoactivated GPCR rhodopsin is terminated by binding of squid arrestin (sArr). sArr forms a light-dependent, high-affinity complex with squid rhodopsin, which does not require prior receptor phosphorylation for interaction. This is at odds with classical mammalian GPCR desensitization where an agonist-bound phosphorylated receptor is needed to break stabilizing constraints within arrestins, the so-called “three-element interaction” and “polar core” network, before a stable receptor–arrestin complex can be established. Biophysical and mass spectrometric analysis of the squid rhodopsin–arrestin complex indicates that in contrast to mammalian arrestins, the sArr C-tail is not involved in a stable three-element interaction. We determined the crystal structure of C-terminally truncated sArr that adopts a basal conformation common to arrestins and is stabilized by a series of weak but novel polar core interactions. Unlike mammalian arrestin-1, deletion of the sArr C-tail does not influence kinetic properties of complex formation of sArr with the receptor. Hydrogen–deuterium exchange studies revealed the footprint of the light-activated rhodopsin on sArr. Furthermore, double electron–electron resonance spectroscopy experiments provide evidence that receptor-bound sArr adopts a conformation different from the one known for arrestin-1 and molecular dynamics simulations reveal the residues that account for the weak three-element interaction. Insights gleaned from studying this system add to our general understanding of GPCR–arrestin interaction.

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

Proteasomes are key protease complexes responsible for protein degradation, and their localization changes with the growth conditions. This work in yeast shows that proteasomes exit the nucleus with the transition from proliferation to quiescence. Ubiquitin is a key player in proteasome dynamics and cytoplasmic proteasome granule formation., Proteasomes are essential for protein degradation in proliferating cells. Little is known about proteasome functions in quiescent cells. In nondividing yeast, a eukaryotic model of quiescence, proteasomes are depleted from the nucleus and accumulate in motile cytosolic granules termed proteasome storage granules (PSGs). PSGs enhance resistance to genotoxic stress and confer fitness during aging. Upon exit from quiescence PSGs dissolve, and proteasomes are rapidly delivered into the nucleus. To identify key players in PSG organization, we performed high-throughput imaging of green fluorescent protein (GFP)-labeled proteasomes in the yeast null-mutant collection. Mutants with reduced levels of ubiquitin are impaired in PSG formation. Colocalization studies of PSGs with proteins of the yeast GFP collection, mass spectrometry, and direct stochastic optical reconstitution microscopy of cross-linked PSGs revealed that PSGs are densely packed with proteasomes and contain ubiquitin but no polyubiquitin chains. Our results provide insight into proteasome dynamics between proliferating and quiescent yeast in response to cellular requirements for ubiquitin-dependent degradation.

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2- bis -tetrahydrofuranyl urethane ( bis -THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC 50 s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIV A02 ) compared to ritonavir (RTV; EC 50 , >1.0 μM) and tipranavir (TPV; EC 50 , 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIV DRV R P20 ), with a 2.6-fold increase in its EC 50 (0.097 μM) compared to its corresponding EC 50 (0.038 μM) against wild-type HIV-1 NL4-3 (HIV WT ). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30′ of HIV A02 protease (PR A02 ) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30′ due to an absence of polar groups. The P2′ thiazolyl moiety of RTV showed two conformations in the crystal structure of the PR A02 -RTV complex, one of which showed loss of contacts in the S2′ binding pocket of PR A02 , supporting RTV's compromised antiviral activity (EC 50 , >1 μM). Thus, the conserved H-bonding network of P2- bis -THF-containing GRL008 with the backbone of G27, D29, D30, and D30′ most likely contributes to its persistently greater antiviral activity against HIV WT , HIV A02 , and HIV DRV R P20 .

Crystal structure of multidrug-resistant (MDR) clinical isolate 769, human immunodeficiency virus type-1 (HIV-1) protease in complex with lopinavir (LPV) (PDB ID: 1RV7) showed altered binding orientation of LPV in the expanded active site cavity, causing loss of contacts and decrease in potency. In the current study, with a goal to restore the lost contacts, three libraries of LPV analogs containing extended P1 and/or P1' phenyl groups were designed and docked into the expanded active site cavity of the MDR769 HIV-1 protease. The compounds were then ranked based on three criteria: binding affinity, overall binding profile and predicted pharmacological properties. Among the twelve proposed extensions in different combinations, compound 14 (consists of para-fluoro phenyl group as both P1 and P1' moieties) was identified as a lead with improved binding profile, binding affinity against the MDR protease and favorable predicted pharmacological properties comparable to those of LPV. The binding affinity of 14 against wild type (NL4-3) HIV-1 protease was comparable to that of LPV and was better than LPV against an ensemble of MDR HIV-1 protease variants. Thus, 14 shows enhanced binding affinity by restoring lost contacts in the expanded active site cavity of MDR769 HIV-1 protease variants suggesting that it may have higher potency compared to that of LPV and hence should be further synthesized and evaluated against NL4-3 as well as MDR variants of HIV-1.

Lopinavir (LPV) is a second generation HIV-1 protease inhibitor. Drug resistance has rapidly emerged against LPV since its US FDA approval on September 15, 2000. Mutations at residues 32I, L33F, 46I, 47A, I54V, V82A, I84V, and L90M render the protease drug resistant against LPV. We report the crystal structure of a clinical isolate multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) complexed with LPV and the in vitro enzymatic IC50 of LPV against MDR 769. The structural and functional studies demonstrate significant drug resistance of MDR 769 against LPV, arising from reduced interactions between LPV and the protease target.

Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC(50) of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTV and MDR HIV-1 protease.

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence. [Formula: see text].

We report here that GRL-10413, a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a hydroxyethylamine sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50] of 0.00035 to 0.0018 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC50] = 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1NL4-3 variants selected by use of atazanavir, lopinavir, or amprenavir (APV) at concentrations of up to 5 μM (EC50 = 0.0021 to 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that against APV. In addition, GRL-10413 showed favorable central nervous system (CNS) penetration properties as assessed with an in vitro blood-brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants, with favorable CNS penetration capability, and that the newly modified P1 moiety may confer desirable features in designing novel anti-HIV-1 PIs.

We identified GRL-1388 and -1398, potent nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing a bicyclic P2 functional moiety, tetrahydropyrano-tetrahydrofuran (Tp-THF). GRL-1388 was as potent as darunavir (DRV) against various drug-resistant HIV-1 laboratory strains with 50% effective concentration (EC 50 s) of 2.6 to 32.6 nM. GRL-1398 was significantly more potent against such variants than DRV with EC 50 s of 0.1 to 5.7 nM. GRL-1388 and -1398 were also potent against multiple-PI-resistant clinical HIV-1 variants ( CL HIV-1 MDR ) with EC 50 s ranging from 2.7 to 21.3 nM and from 0.3 to 4.8 nM, respectively. A highly DRV-resistant HIV-1 variant selected in vitro remained susceptible to GRL-1398 with the EC 50 of 21.9 nM, while the EC 50 of DRV was 214.1 nM. When HIV-1 NL4-3 was selected with GRL-1398, four amino acid substitutions—leucine to phenylalanine at a position 10 (L10F), A28S, L33F, and M46I—emerged, ultimately enabling the virus to replicate in the presence of >1.0 μM the compound beyond 57 weeks of selection. When a mixture of 10 different CL HIV-1 MDR strains was selected, the emergence of resistant variants was more substantially delayed with GRL-1398 than with GRL-1388 and DRV. Modeling analyses revealed that GRL-1398 had greater overall hydrogen bonding and hydrophobic interactions than GRL-1388 and DRV and that GRL-1388 and -1398 had hydrogen bonding interactions with the main chain of the active-site amino acids (Asp29 and Asp30) of protease. The present findings warrant that GRL-1398 be further developed as a potential drug for treating individuals with HIV-1 infection.

Four aspartyl proteases known as plasmepsins are involved in the degradation of hemoglobin by Plasmodium falciparum, which causes a large percentage of malaria deaths. The enzyme plasmepsin II (Plm II) is the most extensively studied of these aspartyl proteases and catalyzes the initial step in the breakdown of hemoglobin by the parasite. Several groups have reported the design, synthesis, and evaluation of reversible peptidomimetic inhibitors of Plm II as potential antimalarial agents. We now report four peptidomimetic analogues, compounds 6-9, which are rationally designed to act as mechanism-based inhibitors of Plm II. Three of these analogues produce potent irreversible inactivation of the enzyme with IC(50) values in the low nanomolar range. Of these three compounds, two retain the low micromolar IC(50) values of the parent compound in Plasmodium falciparum (clone 3D7) infected erythrocytes. These analogues are the first examples of fully characterized mechanism-based inactivators for an aspartyl protease and show promise as novel antimalarial agents.

Children with Down syndrome (DS) with acute megakaryocytic leukemia (AMkL) have very high survival rates compared with non-DS AMkL patients. Somatic mutations identified in the X-linked transcription factor gene, GATA1, in essentially all DS AMkL cases result in the synthesis of a shorter (40 kDa) protein (GATA1s) with altered transactivation activity and may lead to altered expression of GATA1 target genes. Using the Affymetrix U133A microarray chip, we identified 551 differentially expressed genes between DS and non-DS AMkL samples. Transcripts for the bone marrow stromal-cell antigen 2 (BST2) gene, encoding a transmembrane glycoprotein potentially involved in interactions between leukemia cells and bone marrow stromal cells, were 7.3-fold higher (validated by real-time polymerase chain reaction) in the non-DS compared with the DS group. Additional studies confirmed GATA1 protein binding and transactivation of the BST2 promoter; however, stimulation of BST2 promoter activity by GATA1s was substantially reduced compared with the full-length GATA1. CMK sublines, transfected with the BST2 cDNA and incubated with HS-5 bone marrow stromal cells, exhibited up to 1.7-fold reduced cytosine arabinoside (ara-C)-induced apoptosis, compared with mock-transfected cells. Our results demonstrate that genes that account for differences in survival between DS and non-DS AMkL cases may be identified by microarray analysis and that differential gene expression may reflect relative transactivation capacities of the GATA1s and full-length GATA1 proteins.

Human immunodeficiency virus type − 1 (HIV-1) protease, a homodimeric aspartyl protease, is a critical drug target in designing anti-retroviral drugs to treat HIV/AIDS. Multidrug-resistant (MDR) clinical isolate-769 HIV-1 protease (PDB ID: 3PJ6) has been shown to exhibit expanded active site cavity with wide-open conformation of flaps (Gly48 – Gly52) due to the accumulation of multiple mutations. In this study, an HIV-1 protease dimerization inhibitor (PDI) − TLF-PafF, was evaluated against MDR769 HIV-1 protease using X-ray crystallography. It was hypothesized that co-crystallization of MDR769 HIV-1 protease in complex with TLF-PafF would yield either a monomeric or a disrupted dimeric structure. However, crystal structure of MDR769 I10V HIV-1 protease co-crystallized with TLF-PafF revealed an undisrupted dimeric protease structure (PDB ID: 4NKK) that is comparable to the crystal structure of its corresponding apo-protease (PDB ID: 3PJ6). In order to understand the binding profile of TLF-PafF as a PDI, docking analysis was performed using monomeric protease (prepared from the dimeric crystal structure, PDB ID: 4NKK) as docking receptor. Docking analysis revealed that TLF-PafF binds at the N and C termini (dimerization domain) in a clamp shape for the monomeric wild type receptor but not the MDR769 monomeric receptor. TLF-PafF preferentially showed higher binding affinity to the expanded active site cavity of MDR769 HIV-1 protease than to the termini. Irrespective of binding location, the binding affinity of TLF-PafF against wild type receptor (-6.7 kcal/mol.) was found to be higher compared to its corresponding binding affinity against MDR receptor (-4.6 kcal/mol.) suggesting that the MDR769 HIV-1 protease could be resistant to the PDI-activity of TLF-PafF, thus supporting the dimeric crystal structure (PDB ID: 4NKK).

GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3( R ),3a( S ),6a( R )- bis -tetrahydrofuranylurethane ( bis -THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2′ moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PR WT ). Both GRL007 ( K i of 12.7 pM with PR WT ) and GRL008 ( K i of 8.9 pM) inhibited PR WT with high potency in vitro . X-ray crystallographic analysis of PR WT in complex with GRL007 or GRL008 showed that the bis -THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PR WT . The P2′ moiety of both compounds showed one direct contact with the backbone of Asp30′ and a bridging polar contact with Gly48′ through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC 50 ] of >1 μM) while GRL008 was highly active (EC 50 of 0.04 μM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2′ benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2′ moiety may improve its antiretroviral features.

Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: 1TW7), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC50: 4.4nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6a against both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of (15)N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

Two potent inhibitors (compounds 1 and 2) of malarial aspartyl protease, plasmepsin-II, were evaluated against wild type (NL4-3) and multidrug-resistant clinical isolate 769 (MDR) variants of human immunodeficiency virus type-1 (HIV-1) aspartyl protease. Enzyme inhibition assays showed that both 1 and 2 have better potency against NL4-3 than against MDR protease. Crystal structures of MDR protease in complex with 1 and 2 were solved and analyzed. Crystallographic analysis revealed that the MDR protease exhibits a typical wide-open conformation of the flaps (Gly48 to Gly52) causing an overall expansion in the active site cavity, which, in turn caused unstable binding of the inhibitors. Due to the expansion of the active site cavity, both compounds showed loss of direct contacts with the MDR protease compared to the docking models of NL4-3. Multiple water molecules showed a rich network of hydrogen bonds contributing to the stability of the ligand binding in the distorted binding pockets of the MDR protease in both crystal structures. Docking analysis of 1 and 2 showed a decrease in the binding affinity for both compounds against MDR supporting our structure–function studies. Thus, compounds 1 and 2 show promising inhibitory activity against HIV-1 protease variants and hence are good candidates for further development to enhance their potency against NL4-3 as well as MDR HIV-1 protease variants.

Charcot-Marie-Tooth disease (CMT), a hereditary motor and sensory neuropathy, is the most common genetic neuropathy with an incidence of 1 in 2600. Several forms of CMT have been identified arising from different genomic abnormalities such as CMT1 including CMT1A, CMT1B, and CMTX. CMT1 with associated peripheral nervous system (PNS) demyelination, the most frequent diagnosis, demonstrates slowed nerve conduction velocities and segmental demyelination upon nerve biopsy. One of its subtypes, CMT1A, presents a 1.5-Mb duplication in the p11-p12 region of the human chromosome 17 which encodes peripheral myelin protein 22 (PMP22). CMT1B, a less common form, arises from the mutations in the myelin protein zero (MPZ) gene on chromosome 1, region q22-q23, which encodes the major structural component of the peripheral myelin. A rare type of CMT1 has been found recently and is caused by point mutations in early growth response gene 2 (EGR2), encoding a zinc finger transcription factor in Schwann cells. In addition, CMTX, an X-linked form of CMT, arises from a mutation in the connexin-32 gene. Myelin protein zero, associated with CMT1B, is a transmembrane protein of 219 amino acid residues. Human MPZ consists of three domains: 125 residues constitute the glycosylated immunoglobulin-like extracellular domain; 27 residues span more » the membrane; and 67 residues comprise the highly basic intracellular domain. MPZ makes up approximately 50% of the protein content of myelin, and is expressed predominantly in Schwann cells, the myelinating cell of the PNS. Myelin protein zero, a homophilic adhesion molecule, is a member of the immunoglobulin super-family and is essential for normal myelin structure and function. In addition, MPZ knockout mice displayed abnormal myelin that severely affects the myelination pathway, and overexpression of MPZ causes congenital hypomyelination of peripheral nerves. Myelin protein zero mutations account for {approx}5% of patients with CMT. To date, over 125 different mutations in the MPZ gene leading to peripheral neuropathy in patients have been reported worldwide (http://www.molgen. ua.ac.be/CMTMutations). All identified mutations resulting in a change or deletion of amino acid residues in MPZ give rise to neuropathy with the exception of R215L, which instead causes a benign polymorphism. Furthermore, more detailed analysis has classified the MPZ mutations into two major groups. In the first group, the mutations disrupt the intracellular processing of MPZ and are primarily associated with early onset neuropathy. It has been proposed that the mutated MPZ is trapped inside the cell rather than being transported to the plasma membrane. However, other evidence suggests that the mutated MPZ protein is expressed on the plasma membrane, but dominant-negatively disrupts the structure of myelin. In the second group, the MPZ mutations are associated with late onset neuropathy as these mutations cause only mild demyelination. The underlying mechanism is elusive with the hypothesis being that the second group of mutations cause minor abnormalities in the myelin sheath that over time may lead to aberrant Schwann cell-axon interactions and subsequently to axonal degeneration. The crystal structure of the extracellular domain of human MPZ (hP0ex) fused with maltose binding protein (MBP) is reported at 2.1 {angstrom} resolution. While the crystal structure of rat MPZ extracellular domain (rP0ex) is available, the crystal structure of the human counterpart is useful for the analysis of the two homologs as well as a comparison between the two species. The hP0ex molecule reveals subtle structural variations between two homologs allowing comparison of the human myelin protein zero to that of the rat protein. The alignment of these homologs is shown in Figure 1(a). « less