extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Description of HIV-1 Group M Molecular Epidemiology and Drug Resistance Prevalence in Equatorial Guinea from Migrants in Spain

Background The HIV epidemic is increasing in Equatorial Guinea (GQ), West Central Africa, but few studies have reported its HIV molecular epidemiology. We aimed to describe the HIV-1 group M (HIV-1M) variants and drug-resistance mutations in GQ using sequences sampled in this country and in Spain, a frequent destination of Equatoguinean migrants. Methods We collected 195 HIV-1M pol sequences from Equatoguinean subjects attending Spanish clinics during 1997-2011, and 83 additional sequences sampled in GQ in 1997 and 2008 from GenBank. All (n = 278) were re-classified using phylogeny and tested for drug-resistance mutations. To evaluate the origin of CRF02_AG in GQ, we analyzed 2,562 CRF02_AG sequences and applied Bayesian MCMC inference (BEAST program). Results Most Equatoguinean patients recruited in Spain were women (61.1%) or heterosexuals (87.7%). In the 278 sequences, the variants found were CRF02_AG (47.8%), A (13.7%), B (7.2%), C (5.8%), G (5.4%) and others (20.1%). We found 6 CRF02_AG clusters emerged from 1983.9 to 2002.5 with origin in GQ (5.5 sequences/cluster). Transmitted drug-resistance (TDR) rate among naïve patients attended in Spain (n = 144) was 4.7%: 3.4% for PI (all with M46IL), 1.8% for NRTI (all with M184V) and 0.9% for NNRTI (Y188L). Among pre-treated patients, 9/31 (29%) presented any resistance, mainly affecting NNRTI (27.8%). Conclusions We report a low (<5%) TDR rate among naïve, with PI as the most affected class. Pre-treated patients also showed a low drug-resistance prevalence (29%) maybe related to the insufficient treatment coverage in GQ. CRF02_AG was the prevalent HIV-1M variant and entered GQ through independent introductions at least since the early 1980s.

Introduction
The human immunodeficiency virus type 1 group M (HIV-1M), responsible for the HIV pandemic, originated from a zoonotic transmission from chimpanzees into humans in Cameroon [1]. For decades, the virus evolved and diversified into different lineages in the Congo River basin, where the highest viral diversity circulates [2]. Today, HIV-1M is sub-divided into 9 pure subtypes (A-D, F-H, J and K) and recombinants between them (http:// www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html): 58 circulating recombinant forms (CRFs) and multiple unique recombinant forms (URFs).
Equatorial Guinea (GQ), a Spanish colony until 1968, is a small country located in West Central Africa between Cameroon and Gabon. The estimated number of people living with HIV in GQ (20,000 in 2009) increases annually, and antiretroviral treatment (ART) only reached 48% of them in 2011 [3]. Despite its location, close to the epicenter of HIV-1 pandemic, only two local studies [4,5] have so far reported the HIV molecular epidemiology in GQ. Ortiz [6].
The latter also reported a rate of transmitted antiretroviral drug resistance (TDR) of 4.2%. This rate represents the percentage of naïve patients infected with HIV variants carrying drug resistance mutations, and threatens the effectiveness of ART. Recent studies report an increasing TDR prevalence in low-and middle-income countries following ART scale-up [7]. This rise especially affects sub-Saharan Africa, driven by the resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) [8,9]. The TDR rate in Cameroon is around 8-9% [10,11] and lower than 5% in Gabon [12]. HIV molecular epidemiology studies are crucial in these areas to prevent the widespread transmission of drug resistance, especially those lacking systematic surveillance as GQ.
We aimed to describe the circulating HIV-1M variants and the presence of antiretroviral drug resistance mutations in Equatorial Guinea. To achieve this, we applied phylogenetic methods to a combination of viral sequences sampled in Spain (frequent destination of Equatoguinean migration) and publicly available sequences sampled in the sub-Saharan country.

Study Population
All the HIV-infected subjects coming from Equatorial Guinea with available HIV-1M pol sequences, and attended in Spanish HIV/AIDS clinics (mainly in Madrid) were collected. They were 195 adult and pediatric patients sampled between 1997 and 2011. Most sequences were previously reported [13][14][15][16], while 37 were unpublished. In most cases (136, 69.7%) they included the complete protease (PR, 297nt) and partial reverse transcriptase (RT, 860nt of mean length). In 58 (29.7%) and 1 (0.5%) cases, only the PR or the RT sequence was available, respectively. In addition, all the HIV-1 pol sequences (n = 83) sampled in Equatorial Guinea and available in the GenBank (www.ncbi. nlm.nih.gov/genbank) were retrieved. They belonged to two sequences batches sampled in 1997 (not published) and 2008 [5]. GenBank accession numbers of the sequences included are provided in Accession S1.

HIV-1M Subtyping
HIV-1M variants were re-classified by phylogenetic analysis of the 278 pol sequences, including recombinants not available at publishing time. Representative sequences of the HIV-1M 9 subtypes and 49 CRFs downloaded from Los Alamos HIV sequence database (LANL-DB, http://www.hiv.lanl.gov) were used as references. Sequences were aligned manually, and initial neighbor-joining trees were built using MEGA5 (http://www. megasoftware.net) under the Kimura two-parameter model with 1,000 bootstrap re-sampling. Subtypes were further confirmed in approximately maximum-likelihood (ML) phylogenetic trees constructed under the general time reversible model of nucleotide substitutions with gamma-distributed heterogeneity rate (GTR+C) using FastTree v2.1.3 (http://www.microbesonline.org/fasttree). The topology robustness was tested by likelihood-based local branch support. In sequences not ascribed to any known subtype or CRF, recombination analyses were performed using SimPlot v3.5.1 and Recombination Detection Program (RDP, v3alpha44).
In the CRF02_AG subset, we searched monophyletic clusters (associations of $3 samples) that involved only sequences from GQ in ML trees that included all CRF02_AG sequences from LANL-DB (n = 2,652) constructed as described above with FastTree v2.1.3.
The time to the most recent common ancestor (TMRCA) of these Equatoguinean CRF02_AG clusters was estimated using Bayesian Markov chain Monte Carlo (MCMC) inference with the program BEAST v1.6.1 (http://beast.bio.ed.ac.uk). A lognormal distributed prior was placed on the estimated number of nucleotide substitution per site per year (mean = 2.5 6 10 23 ; standard deviation = 0.5). The MCMC chain ran for 10 8 generations, sampling estimates every 10,000th generation. The uncorrelated lognormal molecular clock model and the SRD06 nucleotide substitution were selected. These parameters were chosen based on previous estimates for CRF02_AG datasets [17]. The tree samples were used to generate a maximum clade credibility (MCC) tree, after a 10% burn-in, using TreeAnnotator v1.6.1. Those Equatoguinean CRF02_AG clusters with a high clade support in both ML and Bayesian trees ($95%) were chosen as definitive.

Drug Resistance
For HIV sequences from antiretroviral-naïve patients, the TDR prevalence was defined following the World Health Organization mutation list [18] using the Calibrated Population Resistance tool (http://cpr.stanford.edu). For antiretroviral-exposed patients, substitutions considered in the 2011 International AIDS Society-USA (IAS-USA) mutation list [19] were manually identified.

Ethics Statement
This study was part of a project approved by the review board of the Hospital Ramón y Cajal Clinical Research Ethical Committee. It was designed to protect the rights of all subjects involved under the appropriate local regulations. To maintain subject confidentiality, a unique ID number was assigned to each specimen, and written consent obtained for each patient by clinicians. For the minors involved in the study, written consent was provided by their parents or legal representatives.

Equatoguinean HIV-infected Patients in Spain
The main characteristics of these patients are shown in Table 1.
Most subjects (61.1%) of this cohort were women, and were infected with HIV due to heterosexual risk practices, in concordance with the general situation in sub-Saharan Africa [20]. Sorting patients according to the sample periods shown in Table 1 Table 2 shows the distribution of HIV-1M variants among the 278 pol sequences from Equatoguinean patients, where the recombinant CRF02_AG (47.8%) was predominant. Pure subtypes infected 122 (43.9%) patients, and other CRFs were found in 19 (6.8%) cases. Four sequences were classified as URF (URF_JU, URF_BH, URF_02D and URF_BG) using RDP and SimPlot. Of note, the frequency of subtype B was significantly higher in Equatoguinean subjects sampled in Spain than in those sampled in EQ (9.7% vs. 1.2%, P = 0.012).

HIV-1M Variants Circulating in Equatorial Guinea
The new analysis of the sequences permitted to update their classification, especially among recombinants. We re-classified as CRF02_AG 21 sequences initially assigned to subtype G when they were sampled [21,22], 1 CRF11_cpx reported as subtype J [22], 1 CRF13_cpx and 1 CRF18_cpx also reported as subtype G [21] and 1 CRF22_01A1 described as subtype A [16]. The recombination analyses revealed that 2 sequences isolated in 1999 and 2000 and reported as pure subtypes G and J [21] were URF_BG and URF_JU, respectively.

CRF02_AG Cluster Analysis
In the general ML tree that included the 133 CRF02_AG sequences of the study population and 2,652 worldwide CRF02_AG sequences (Figure 1), we found 7 monophyletic clusters including exclusively Equatoguinean CRF02_AG sequences with .95% of statistical support. The geographic origin of the 2,652 CRF02_AG sequences from LANL-DB is described in the legend of Figure 1. The further analysis with BEAST included a 92 sequence alignment: the CRF02_AG involved in the putative Equatoguinean clusters (n = 39), their closest relatives among the CRF02_AG from LANL-DB according to the ML tree (n = 15), and reference CRF02_AG sequences with diverse sampling dates (n = 38) in order to include at least one sequence per year from 1990 when available. The sampling times of the sequence dataset included in the BEAST analysis are shown in the supplementary Table S1.
The Bayesian MCC tree ( Figure S1) showed that 6 of the 7 putative clusters were supported by a posterior probability of $0.95 (Table 3). Therefore, these 6 clusters were considered as definitive due to their high statistical support. They included 33 Equatoguinean sequences (mean = 5.5 sequences/cluster). All but one (cluster VI) clusters included sequences sampled in both Spain and GQ. The dates of their most recent common ancestors (MRCA) ranged from 1983.9 to 2002.5. Five of the 6 clusters were included among sequences sampled in Western Africa, and one (cluster V) was related to sequences sampled in Central Africa ( Figure 1).

HIV-1M Drug Resistance
Among the 195 sequences collected in Spain from Equatoguinean subjects, 148 (75.9%) corresponded to antiretroviral-naïve patients and 31 (15.9%) to treatment-experienced subjects, with unknown treatment experience in 16 (8.2%) patients. We found at least one transmitted drug-resistance mutation (TDR) in 7 of the 148 (4.7%) naïve patients following the World Health Organization mutations list [18]. TDR was 6.7% (6/90) in women vs. 1.8% (1/56) in men (P = 0.2). Table 4 shows the TDR rate according to the antiretroviral class affected. In 5 (3.4%) subjects, the mutations affected PR inhibitors (PI), with M46I/L substitution in all cases. Another 2 (1.8%) presented the M184V mutation to nucleoside reverse transcriptase inhibitors (NRTI). One of the latter also presented a drug-resistance mutation to non-nucleoside reverse transcriptase inhibitors (NNRTI). No triple-class resistance was found. Among the 31 ART-experienced patients, 9 (29%) presented any drug-resistance mutation according to the IAS-USA list [19]. The highest rate (27.8%) was found for NNRTIresistance due to mutation K103N in 3 of the 7 cases. The PIresistance mutations found were M46L (4 cases), and L90M and I84V (3 cases each). For NRTI-resistance, the mutation M184V was found in all 3 cases. We observed a fluctuation in TDR rates in the three periods shown at Table 1 Table 5 shows the main epidemiological and virological features of the 7 drug-naïve and the 9 drug-experienced patients carrying viruses harboring resistance mutations. Of note, most  naïve (6/7; 85.7%) and pre-treated (6/9 cases; 66.7%) patients carrying drug-resistant viruses were adult women. They were mainly infected by CRF02_AG (2/7 and 4/9, respectively) and subtype B (2/7 and 3/9 cases, respectively). Regarding the sequences retrieved from GenBank and obtained in Equatorial Guinea, the antiretroviral status was only available for the 41 samples described in Djoko et al., which were obtained from naïve HIV-infected population in Malabo. Among them, TDR was 4.9% according to the WHO mutations list.

Discussion
Despite being located in the River Congo basin, where the HIV epidemics originated [1], only two works [4,5] have provided data about the circulation of different HIV strains and drug resistance in Equatorial Guinea. Here, we expand and complete the information available about the HIV Equatoguinean epidemics combining the use of HIV-1M pol sequences from immigrants in Spain (the main host developed country for Equatoguinean migration) and from patients sampled in Equatorial Guinea. This is the larger study covering transmitted drug resistance in this country and the first one analyzing general HIV-infected population and dating the introduction of CRF02_AG.

CRF02_AG Recombinant is Prevalent in Equatorial Guinea
We confirm that recombinant CRF02_AG, the prevalent HIV-1M variant in Western Africa [6], accounts for a half of infections in Equatorial Guinea ( Table 2). This is also the most frequent Branch colors indicate the origin of the sequences (with the same color code in the map). In blue, sequences from Equatoguinean patients (n = 133). In orange, CRF02_AG sequences from Spain (n = 158). In green, CRF02_AG sequences from Western Africa: Nigeria (n = 391), Mali (n = 235), Senegal (n = 185), Ghana (n = 180), Burkina Faso (n = 97), Benin (n = 77), Togo (n = 58), Liberia (n = 4), the Gambia (n = 2) and Guinea-Bissau (n = 2). In red, CRF02_AG sequences from Central Africa: Cameroon (n = 711), Gabon (n = 72), and Democratic Republic of the Congo (n = 7). In black, CRF02_AG sequences from other regions. The 6 monophyletic clusters from Equatorial Guinea further assessed using BEAST are highlighted in yellow and labeled in accordance to  [23] and pediatric [15] HIVinfected population. The sequences' re-analyses identified CRF02_AG isolates classified as subtype G when they were described using only protease sequences [21]. We also found CRF11_cpx, CRF13_cpx, CRF18_cpx and CRF22_01A1 sequences initially described as pure subtypes [16,22]. These complex variants are found in Spain among immigrants and rarely in autochthonous population [14,23]. Recombinants might be underestimated in studies performed in periods that lacked reference sequences published afterwards. Thus, periodic reassessments of HIV classification with updated CRFs might improve the subtyping. In addition, modern computer tools permit to identify previously unnoticed unique recombinant forms when phylogenetic analyses are inconclusive.

CRF02_AG Introduction in Equatorial Guinea
The Bayesian MCMC analysis using BEAST revealed 6 monophyletic, highly statistically supported clusters of Equatoguinean CRF02_AG sequences. This could indicate that this variant entered Equatorial Guinea through several independent introductions occurred at least since the early 1980s, and not through a single introduction event. A previous study proposed that CRF02_AG was introduced in Equatorial Guinea from Cameroon [24], but our results showed that only one of the 6 fell within Central African clades, being the rest related to Western African clades. In many cases, sequences from both regions were interspersed in the analysis (Figure 1), which suggests a frequent circulation of CRF02_AG between countries, as others have also indicated [25]. In both Western and Central Africa the presence of this recombinant, originated at least in the early 1970s [24], has increased in the last years [6].
On the other hand, the observed clustering could also be explained by specific transmission networks, although the available epidemiological information collected in the patients' clinical records does not point to the presence of any of these networks. In addition, as previously commented, all clusters but one (cluster VI) included both CRF02_AG sequences sampled in Spain and in Equatorial Guinea, which minimizes the chance of a local transmission network. Despite these explanations, this alternative possibility cannot be completely ruled out.

Low Rate of Resistance among HIV-1M-infected Equatoguineans
We observed a TDR rate of 4.7% among the 148 antiretroviralnaïve Equatoguineans attending Spanish HIV/AIDS clinics, a figure close to the limit between low and moderate drug-resistance (5%) according to the World Health Organization [26]. This rate is very similar to that found in the only TDR study performed in Equatorial Guinea (4.9%), which screened military personnel in Malabo [5]. Both are lower than in other sub-Saharan countries, including the neighboring Cameroon, with a TDR around 9% [10,11]. Since ART still reaches only 48% of the HIV-infected population in need of therapy in GQ [3], a lower emergence of resistance than in countries with an older ART scale-up is expected [7].
The highest TDR rate was found for PI (3.4%), led by the presence of the substitution M46I/L in all cases. This was also the most frequent PI-resistance mutation among pre-treated patients, and it was previously reported among antiretroviral-naïve military  personnel living in Equatorial Guinea [5]. This high prevalence contrasts with the rare administration of PI in Equatorial Guinea, where the first line treatment includes the NNRTI efavirenz [3]. For transmitted NRTI-resistance, the low presence found (with the predominance of change M184V) agrees with the study of Djoko et al., although they only found the substitution D67N as transmitted NRTI-resistance mutation [5]. Studies in Uganda [7,27] have related the expanded use of NNRTI as peripartum prophylaxis among HIV-infected pregnant women to a higher population-wide prevalence of NNRTIresistance mutations. Thus, the low presence of transmitted NNRTI-resistance mutations in Equatoguineans compared with other sub-Saharan populations [8] could result from the infrequent administration of this prophylaxis in Equatorial Guinea (only in 19% of HIV-infected pregnant women in 2010) [3].
In treated Equatoguinean patients attended in Spain, we found a presence of drug-resistance mutations rate of 29%, with NNRTI as the most affected drug class (27.8%). This lower prevalence than in other sub-Saharan antiretroviral-exposed cohorts [28] could reflect a poor adherence as observed among sub-Saharans living in Spain [29], who present great rates of follow-up losses. This has also been reported in Africa [30], caused by the low accessibility of antiretroviral drugs, as happens in Equatorial Guinea, with an antiretroviral coverage of 48% in 2011. In Equatorial Guinea, the use of antiretroviral drugs started in a regular basis in 2005. Thus, previous sporadic treatments and interrupted exposition to drugs could have caused the appearance of resistance mutations that jeopardizes the success of a future systematic antiretroviral programme. Unfortunately, in most cases the information on specific drug exposure for the pre-treated patients was unavailable, which prevent us from a further interpretation of these data.

HIV Infection Place
The phylogenetic analyses showed that HIV-1M sequences sampled in Spain and/or Equatorial Guinea were interspersed, which would suggest that most of the 195 Equatoguinean patients with clinical follow up in Spain were infected in their country of origin. However, we cannot rule out that some of them could have been infected in Spain. This hypothesis would be supported by the overrepresentation of subtype B (the predominant variant in Spain [23] but infrequent in Central Africa) among samples taken in this host country versus those taken in Equatorial Guinea; and by the higher prevalence of transmitted PI-resistance in these subtype Binfected Equatoguineans sampled in Spain (where treatment is universally available) than in other variants of the study cohort.
Conversely, an evidence for an infection happened in Africa would be the different mutation pattern found in Equatoguineans sampled in Spain than in a study of general, naïve patients [14] (autochthonous and African and non-African foreigners) living in Spain between 1996 and 2010. Thus, the PI-resistance mutation M46L, present in all subtype B viruses with TDR ( Table 5), was rare (,1%) among the subtype B-infected population living in Spain, where L90M was predominant. In addition, if the Equatoguinean patients had been infected in Spain, higher rates of NRTI-and, especially, NNRTI-resistance would have been expected.
In conclusion, the rising HIV drug-resistance transmission in sub-Saharan Africa following antiretroviral rollout highlights the need of periodical surveillance studies to monitor and prevent the resistance emergence, essential information to design or continue the implementation of ART programmes. These studies should be performed among infected people living in the study region. However, in countries lacking surveillance reports (such as Equatorial Guinea), our approach describing the HIV molecular epidemiology of low-income regions from a developed host country can provide any knowledge about the HIV epidemic in specific areas. Figure S1 Maximum clade credibility tree of the 92 CRF02_AG pol sequences included in the BEAST analysis. The definitive clusters are highlighted in yellow and labeled in accordance to Table 3. The asterisk indicates nodes supported by a posterior probability of $0.95. The horizontal axis is expressed in calendar years. Branch colors indicate the origin of the sequences: blue, sequences from Equatoguinean patients (n = 39); green, sequences from Western Africa (n = 21); red, sequences from Central Africa (n = 17); black, other regions (n = 15).


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Resistin in Rodents and Humans

Obesity is characterized by excess accumulation of lipids in adipose tissue and other organs, and chronic inflammation associated with insulin resistance and an increased risk of type 2 diabetes. Obesity, type 2 diabetes, and cardiovascular diseases are major health concerns. Resistin was first discovered as an adipose-secreted hormone (adipokine) linked to obesity and insulin resistance in rodents. Adipocyte-derived resistin is increased in obese rodents and strongly related to insulin resistance. However, in contrast to rodents, resistin is expressed and secreted from macrophages in humans and is increased in inflammatory conditions. Some studies have also suggested an association between increased resistin levels and insulin resistance, diabetes and cardiovascular disease. Genetic studies have provided additional evidence for a role of resistin in insulin resistance and inflammation. Resistin appears to mediate the pathogenesis of atherosclerosis by promoting endothelial dysfunction, vascular smooth muscle cell proliferation, arterial inflammation, and formation of foam cells. Indeed, resistin is predictive of atherosclerosis and poor clinical outcomes in patients with coronary artery disease and ischemic stroke. There is also growing evidence that elevated resistin is associated with the development of heart failure. This review will focus on the biology of resistin in rodents and humans, and evidence linking resistin with type 2 diabetes, atherosclerosis, and cardiovascular disease.

INTRODUCTION
The prevalence of obesity has increased worldwide and is associated with increased risk of type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD), sleep apnea, cancer, fatty liver, and other diseases. It has become clear that obesity promotes a state of chronic inflammation in white adipose tissue (WAT), which leads to insulin resistance. Obesity results in excessive lipid accumulation in adipocytes and macrophages in WAT, which plays an active role in the development of insulin resistance through the release of free fatty acids, proinflammatory cytokines, and adipokines [1]. Adipose tissue secretes adipokines that play major roles in metabolism, immunity, and inflammation. Resistin is secreted primarily by adipocytes in rodents and was initially proposed as a link between obesity and insulin resistance [2]. In humans, resistin is mainly secreted by macrophages [3], suggesting that resistin is linked to inflammation. Obesity, T2DM, and CVD have been recently recognized as chronic inflammatory disorders which may be connected to proinflammatory cytokines as well as adipokines such as resistin [4]. In this review, we will discuss the biology of resistin in rodents and humans, and focus on the evolving roles of resistin in atherosclerosis and CVD in humans.
sistin is primarily expressed in mature white adipocytes. Mouse resistin expression is reduced in 3T3-L1 adipocytes by TZD drugs [5,6] and insulin [5,7]. In contrast, high glucose up-regulates resistin expression in adipocytes [5,8]. There were also conflicting results showing insulin can stimulate resistin secretion in mouse adipocytes [9][10][11]. Resistin expression in mouse adipocytes is suppressed by inflammatory cytokines, e.g., tumor necrosis factor α (TNF-α), [5,12]. Unlike rodents, human resistin is expressed in macrophages and is induced by TNF-α [13,14]. The lack of human resistin expression in adipocytes may be due to loss of a genomic binding site for the nuclear receptor peroxisome proliferator-activated receptor γ that normally controls resistin gene (retn) expression in mouse adipocytes [15]. Dietary intake has a significant effect on the regulation of resistin in rodents. The expression of resistin is decreased in the adipose tissue during fasting, and tends to increase upon refeeding in mice [2,8,16]. Serum resistin is also reduced in fasted mice. Diet-induced obese mice have elevated circulating resistin levels, however, resistin mRNA expression is suppressed in adipose tissue in these mice. Similarly, serum resistin is increased in ob/ob mice while resistin mRNA expression is reduced [16,17].
Resistin circulates in two distinct forms in mice. A disulfidelinked hexamer of resistin is the major form, but a smaller trimeric protein is also detected. The low molecular weight form of trimer is shown to be more bioactive, significantly decreasing hepatic insulin sensitivity during pancreatic clamp study [18]. However, oligomerization is required for the inhibitory action of resistin on glucose uptake in mouse cardiomyocytes [19]. In humans, there are also different molecular isoforms of circulating resistin such as trimeric and oligomeric forms, and the oligomeric form of human resistin is considered to have a more potent effect on the stimulation of proinflammatory cytokines [20][21][22].

RESISTIN IN GLUCOSE HOMEOSTASIS IN RODENTS
Several lines of evidence support a role of resistin in glucose metabolism in rodents. Circulating levels of resistin were higher in obese mice, and the administration of recombinant resistin in normal mice impaired glucose tolerance and insulin action. Furthermore, neutralization of resistin with antiresistin antibody improved insulin sensitivity in diet-induced obese mice [2]. Both central and peripheral administration of recom-binant resistin, or transgenic overexpression of resistin induced hepatic insulin resistance in mice [2,[23][24][25]. Conversely, knockdown or deletion of resistin increased hepatic insulin sensitivity in mice on high-fat diet, and in muscle and WAT in ob/ob mice, leading to a decrease in glucose production and an increase in glucose uptake from peripheral tissue [26,27]. Resistin knockout mice showed low glucose levels after fasting, associated with a decrease in expression of gluconeogenic enzymes in liver [26]. Treatment of 3T3-L1 adipocytes, murine cardiomyocytes, and cultured skeletal muscle cells with murine resistin decreased insulin-stimulated glucose uptake into the cells [2,19,28,29]. All of these findings suggest resistin plays an important role in insulin sensitivity in rodents.
Nevertheless, the resistin receptor or the intracellular signaling pathways of resistin have not yet been clearly identified. Recent studies proposed an isoform of decorin or tyrosine kinase-like orphan receptor 1 as functional resistin receptors which may regulate WAT expansion or modulate glucose homeostasis in rodents [30,31]. Murine resistin has been demonstrated to decrease the phosphorylation of AMP-activated kinase (AMPK) in liver, skeletal muscle, and WAT [24,26,32]. Inhibition of resistin, conversely, led to an increase in the phosphorylation of AMPK which is involved in glucose homeostasis. Resistin interferes with multiple steps involved in the insulin signaling cascade, including phosphorylation of insulin receptor substrates, activation of phosphatidylinositol-3-kinase (PI3K) and protein kinase B/Akt in liver, muscle, and WAT [24,32,33]. Resistin treatment in rodents induces the expression of suppressor of cytokine signaling-3, a known inhibitor of insulin signaling, in liver, muscle, and WAT [17,27,[33][34][35]. Considering adipocyte-derived resistin is linked to insulin resistance in rodents, the potential that rodent data may be applicable to human biology has led to much scientific research.

HUMAN RESISTIN IN OBESITY, INSULIN RESISTANCE AND DIABETES
Resistin is predominantly expressed in peripheral blood mononuclear cells (PBMCs), and its expression increases as the cells differentiate into macrophages in humans [3,14,36]. As in murine resistin, TZD drugs down-regulated the expression of human resistin in macrophages or reduced serum resistin levels [3,14,37]. Resistin expression is also found in the nonadipocyte stromal vascular fraction in WAT, fibrotic liver, and atherosclerotic lesions, indicating that macrophages are the main Diabetes Metab J 2013;37:404-414 http://e-dmj.org source of resistin in humans [38][39][40][41]. Indeed, obese individuals who are likely to have greater infiltration of macrophages in adipose tissue showed increased expression of resistin in adipose tissue samples or had higher circulating levels of resistin than in lean individuals [36,42]. Studies of obese subjects undergoing weight reduction through diet or bariatric surgery have shown inconsistent results. Serum resistin levels were reduced, or not changed, despite weight loss in obese subjects [43][44][45]. There were also several reports that serum resistin levels are not associated with the parameters of obesity or insulin resistance [46][47][48]. However, resistin levels have been associated with visceral, intrathoracic, and pericardial fat in a cross-sectional study [49]. Given the close relationship of murine resistin and insulin resistance, numerous clinical studies have examined a possible relationship of resistin and insulin resistance in obese people with or without diabetes. Since obesity and T2DM are both associated with chronic inflammation in WAT, and resistin is mainly produced by macrophages in humans, it is possible that hyperresistinemia is a contributing factor to these pathophysiological states. However, studies relating human resistin to insulin resistance, or T2DM have shown conflicting results [35,46,47,[50][51][52]. Qatanani et al. [53] generated transgenic mice expressing human resistin via a macrophage promoter and bred them with resistin knockout mice. The humanized resistin mice developed WAT inflammation and insulin resistance, demonstrating that human resistin may link inflammatory responses and glucose homeostasis. This is consistent with prospective case-control studies showing that people with elevated baseline levels of resistin have significantly increased risk of developing T2DM even after adjusting for other risk factors [54,55].

RESISTIN POLYMORPHISMS IN INSULIN RESISTANCE AND ATHEROSCLEROSIS
Epidemiological studies also suggest that resistin plays a role in insulin resistance and T2DM in humans. Several single-nucleotide polymorphisms (SNPs) have been demonstrated to be associated with elevated resistin levels, and up to two-thirds of the variation of serum resistin levels may be attributable to genetic factors [56]. The -638 G>A, -420 C>G, and -358 G>A polymorphisms in the promoter region of human resistin gene (RETN) were associated with resistin levels in Japanese obese individuals [57]. In a Japanese population, the G/G genotype at SNP -420 in RETN was associated with susceptibility to T2DM and also was correlated with monocyte resistin expression and with increased serum resistin levels [58,59]. Moreover, a crosssectional analysis of 2,078 Japanese subjects showed that plasma resistin was associated with SNP -420 and also correlated with insulin resistance [60]. The -420G and -537A alleles were associated with increased resistin levels but not with T2DM in a Korean population [61]. In the 5-year prospective study from a Chinese subjects, both -420G and +62A alleles were significant predictors of glycemic progression [62]. Although -420 C>G SNP associated with high resistin levels appears to be related to T2DM in many studies of Asian populations, the data in Caucasian populations have been conflicting [55]. An Italian study showed that the presence of -420 C/G SNP in RETN is associated with increased obesity and metabolic syndrome but not with resistin levels [63]. An American cohort of Caucasian nondiabetic subjects has shown an association of -420G allele with resistin level, but no association with insulin resistance [64]. In the Framingham Offspring Study cohort of 2,531 participants, it has been shown that not -420 C>G SNP but SNPs in the 3' region of RETN are associated with resistin levels [65]. However, El-Shal et al. [66] have recently demonstrated that both -420 C>G and +299 G>A SNP were significantly associated with resistin level, obesity, and insulin resistance in Egyptian obese subjects.
The role of human resistin in the pathogenesis of atherosclerosis is also gaining research attention. Tang et al. [67] have shown that -420 C/G SNP in RETN is associated with an increased risk of coronary artery disease (CAD) in a Chinese population. In contrast, a couple of studies of the -420 variant in Europeans and Caucasians found no correlation with carotid and coronary atherosclerosis, respectively [63,64]. The discrepancy in the results among populations might be attributable to ethnic differences.
It is noteworthy that numerous inflammatory factors upregulated by human resistin are known to be involved in the development of insulin resistance. Therefore, it is possible that resistin induced by inflammation may play a role in the development of insulin resistance in humans. To address this issue, Park et al. [81] generated mice lacking murine resistin but transgenic for a bacterial artificial chromosome containing human resistin (BAC-Retn), whose expression was similar to levels in humans. LPS increased serum resistin levels in this model, and resulted in mild hypoglycemia compared with mice lacking murine and human resistin. In addition, the BAC-Retn mice developed hepatic insulin resistance under chronic endotoxemia, accompanied by inflammation in liver and skeletal muscle, supporting the role of resistin in the pathophysiology of inflammation-induced insulin resistance in humans [81]. A recent study suggested that human resistin competes with LPS for binding to Toll-like receptor 4 (TLR4) which could mediate some of the proinflammatory effects of resistin [82], however, it is unknown whether TLR4 is the predominant resistin receptor in humans.

HUMAN RESISTIN IN ATHEROSCLEROSIS
Given the evidence that resistin is found in human atherosclerotic lesions and affects vascular endothelial function [40,41,83], it is possible resistin is involved in the development of athero-sclerosis and CVD. Treatment of human resistin increased proliferation and migration of human endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Cell proliferation and migration induced by resistin in both EC and VSMC appears to be mediated through the extracellular signal-regulated kinase, PI3K, or p38 mitogen-activated protein kinase signaling pathways [84][85][86]. Resistin was also shown to attenuate the ability of insulin signaling, inhibit endothelial nitric-oxide synthase, and increase oxidative stress in human aortic and coronary artery EC [86,87]. In addition, resistin up-regulated the expression of adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, P-selectin, and fractalkine, and also increased proinflammatory factors such as MCP-1, PAI-1, endothelin-1, matrix metalloproteinases, and vascular endothelial growth factor receptors, and, thereby, promoted monocyte adhesion in vascular EC [17,41,83,84,88,89]. Furthermore, resistin was expressed in human atheroma samples, and increased lipid accumulation through the up-regulation of CD36 expression and an increase in cholesteryl ester deposition in human macrophages, suggesting a role of resistin as a modulator for macrophage to foam cell transformation [40,41,90,91]. In addition, resistin has been shown to induce a prothrombotic phenotype by upregulating tissue factor expression in human coronary artery EC [92,93]. Taken together, these findings indicate that resistin may contribute to EC dysfunction, VSMC proliferation, arterial inflammation, and accumulation of cholesterol in macrophages, suggesting that resistin plays an important role in the pathogenesis and progression of atherosclerosis in humans.

CLINICAL IMPLICATIONS OF RESISTIN IN CVD
There is the growing evidence that resistin is involved in the pathogenesis of CVD. Reilly et al. [48] showed that elevated resistin levels were predictive of coronary atherosclerosis, independent of CRP, in asymptomatic individuals. Resistin was associated with the presence and severity of CAD in patients undergoing coronary angiography and those with stable CAD [94,95]. Moreover, resistin predicted restenosis after coronary artery stenting and was an independent predictor of major adverse cardiovascular events in patients with CAD [96][97][98][99]. In Korean patients with acute myocardial infarction (MI), resistin was an independent predictor of all cause mortality during the mean follow-up period of 12 months [100]. A European study found that high plasma resistin levels were associated with an increased risk of MI but not with risk of ischemic stroke in healthy subjects, even after adjustment for established cardiovascular risk factors including CRP [101]. In regard to the risk of ischemic stroke, a cross-sectional study from Japan and two nested case-control studies from Europe and United States showed that resistin was significantly associated with ischemic stroke among healthy individuals [102][103][104]. Higher resistin levels were also associated with poorer prognosis in patients with CAD or atherothrombotic stroke [99,[105][106][107]. More recently, an analysis of 2,313 diabetic patients of European ancestry from two cross-sectional and two prospective studies showed that high serum resistin was a risk factor for CVD and all cause mortality in patients with T2DM [108]. Interestingly, resistin was also shown to be associated with vascular inflammation measured using 18F-fluorodeoxyglucose-positron emission tomography among healthy individuals [109]. De-spite some studies showing no association between resistin and CVD [110][111][112], a number of animal and clinical studies are strongly supporting the idea that resistin is a potential mediator of atherosclerosis and CAD in humans.
Several animal studies have investigated the effects of resistin on cardiomyocytes. Murine resistin directly reduced both basal and insulin-stimulated glucose uptake in mouse cardiomyocytes [19]. Murine resistin was shown to be up-regulated by mechanical stretch and the overexpression of resistin depressed cardiac contractility, promoting cardiac hypertrophy in rat cardiomyocytes [113,114], suggesting resistin may affect cardiac function in animal models. Regarding the effects of human resistin on cardiomyocytes, however, very few studies have been reported. Human resistin inhibited insulin-stimulated glucose uptake in isolated mouse cardiomyocytes [19]. Preconditioning with recombinant human resistin prior to ischemia significantly impaired contractile recovery during reperfusion and stimulated cardiac TNF-α secretion in isolat-  ed rat hearts, suggesting human resistin has direct cardiac actions [115]. Emerging evidence suggests that elevated resistin is associated with the development of heart failure (HF). Takeishi et al. [116] have found that resistin is correlated with the severity of HF and predicts adverse cardiac events in patients with established HF. In addition, two large cohort studies have shown that high resistin is independently associated with new-onset HF, even after adjusting for known risk factors [117][118][119]. However, the causal role of resistin in HF is unclear and needs further investigation.

CONCLUSIONS
Resistin was initially described as a link between obesity and insulin resistance in rodents. In rodents, resistin is almost exclusively expressed in white adipocytes, whereas human resistin is predominantly expressed in macrophages. The phenotypes of humanized resistin transgenic mice suggest similar roles of murine and human resistin in insulin resistance. Studies in humanized resistin transgenic mice and epidemiological data also suggest that human resistin is a potential mediator between inflammation, and insulin resistance and atherosclerosis (Fig. 1). The identification of the resistin receptor and specific signaling mechanisms will help address crucial questions concerning the biology of resistin in health and disease. Insights into the pathophysiological role of resistin will facilitate the development of novel diagnostic and treatment tools for diabetes, and inflammatory and CVDs.

CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.


s2fieldsofstudy: Biology Chemistry

text:
Overcoming AC220 resistance of FLT3-ITD by SAR302503

Activating mutations in FLT3 (Fms-like tyrosine kinase 3) by internal tandem duplication (ITD) mutations are found in approximately 30% of patients with acute myeloid leukemia (AML) and are associated with poor outcome in this patient population. Numerous FLT3 inhibitors have been tested for the treatment of AML, but these inhibitors have shown variable responses that were attributed to heterogeneity in AML and mechanistic differences associated with inhibitory mechanism employed by these inhibitors.1 Nevertheless, many patients showed a reduction in blast counts and hematological improvements, but these remissions were not durable that questioned the validity of FLT3 as a therapeutic target in AML. To address this, an elegant translational study designed by Neil Shah's group has identified the AC220 resistant mutations in all FLT3-ITD relapsed patients that definitively demonstrated the validity of FLT3-ITD as a therapeutic target in human AML.2 Furthermore, the emergence of resistant mutations has been reported from the relapsed AML patients with FLT3-ITD treated with PKC4123 and sorafenib.4 In addition, an in vitro resistant screening identified mutation at gatekeeper residue conferred cross-resistance to all known FLT3 inhibitors.5 These observations suggest that secondary mutations conferring resistance in the kinase domain will pose a significant clinical challenge, which prompted us to identify new inhibitors against the FLT3 resistant variants.

SAR302503 is a rationally designed small-molecule ATP-competitive inhibitor of JAK2, and it had been shown to have a high degree of kinase selectivity for JAK2 and FLT3 in in vitro kinase assays.6, 7 Given its equal potency toward FLT3 prompted us to test its efficacy against FLT3-ITD variants resistant to AC220. SAR302503 inhibited proliferation of BAF3 cells expressing FLT3 wild type and FLT3-ITD, with IC50 values of 119 and 330 nM, respectively (Figure 1a), whereas parental BAF3 cells and BAF3-JAK2-V617F cells were inhibited at IC50 values of ∼1100 and 600 nM, respectively (Supplementary Figure 1a). In accordance to this, western blotting of phospho-STAT5 and phospho-FLT3 showed reduced phosphorylation at concentrations that parallel the concentrations required to inhibit cell proliferation (Figure 1c). These observations suggest that SAR302503 is more selective to FLT3-WT>FLT3-ITD>JAK2-V617F, and it can be exploited for therapeutic targeting of FLT3-ITD in AML (Supplementary Figure 1a). Next, we analyzed the activity of SAR302503 against five different kinase domain variants of FLT3-ITD that had been shown to confer resistance against AC220. The growth of BAF3 cells expressing resistant variants of FLT3-ITD was inhibited completely within the range of 800 nM (Figure 1a). Interestingly, two FLT3-ITD variants (D839G and Y842H) were found to be hypersensitive to the drug (Figures 1a and b) while mutations at gatekeeper residue F691L and D835F/Y showed only twofold resistance to the drug (Figure 1b). Next, we performed in vitro resistant screening as described previously,8 to identify patterns of drug resistance using cells expressing FLT3-ITD, FLT3-ITD-F691L and FLT3-ITD-D835Y at 3 μM (Cmax value in human6, 9) of SAR302503. Surprisingly, we could not see the emergence of resistant clones against SAR302503 at this concentration (Supplementary Figure 1b). To rule out any off-target effect, we performed screening of BAF3 cells expressing Tel-JAK3 (having a cellular IC50 value of 2300 nm) at 2000 and 3000 nm of SAR302503, which showed growth of innumerable colonies (Supplementary Figure 1b). These observations suggest that the SAR302503 is a potent inhibitor of FLT3-ITD and is very effective against AC220 resistant variants and prevents the emergence of resistant clones.

Figure 1

SAR302503 can efficiently inhibit the proliferation of native and AC220-resistant variants of FLT3-ITD. (a) Dose–response sigmoidal curve showing the cellular proliferation of BAF3 cells expressing FLT3 and its kinase domain AC220 resistant variants ...

To understand the structural basis of SAR302503-mediated inhibition of FLT3 kinase, we constructed a model of the SAR302503-FLT3 kinase structure using a combination of homology modeling and induced-fit docking.10 We used the crystal structure of FLT3 crystallized in an inactive conformation and with related inhibitor−kinase complexes: for example, ABL complexed with nilotinib in closed and inactive conformation (PDB:3CS9); Ppy-A bound to the active conformation of ABL-T315I (PDB:2Z60); and JAK2 bound to isoquinoline in active conformation (PDB:2B7A). Our in silico analysis predicts that SAR302503 binds to an open and enzymatically active conformation of FLT3, in which residue Phe 830 of the DFG motif is displaced to accommodate the benzensulfonamide ring of the inhibitor (Figure 2). In this model, the pyrimidine ring of SAR302503 makes hydrophobic contacts with Leu 616 and Leu 818. Similar to other ATP-competitive inhibitors, SAR302503 anchors to the ATP site by two hydrogen bonds with residues Cys 694 from the kinase hinge region and Asn 816 from the catalytic loop (Figures 2a and b). Our model suggests that the enhanced sensitivity of SAR302503 for the variants D839G and Y842H is due to the destabilization of the inactive conformation by these mutations that in effect would stabilize the open and active conformation to which the drug preferentially binds, thus conferring hypersensitive response (Figures 2d and e). Given the lack of direct interaction of SAR302503 with resistant variants F691L and D835F/Y, our model suggests that these mutations would destabilize the open and active conformation rather than inactivating the kinase, therefore having sufficient catalytic activity to support cellular transformation. In support of this model, a substitution of Leucine for Phe 691 will weaken the hydrophobic spine that in effect will destabilize the active conformation and may weaken the kinase.11 However, substitution of Asp 835 with phenylalanine will destabilize the activation loop from both active and inactive conformation suggesting that it is stabilizing an intermediate conformational state.12 In inactive conformation, Asp 835 interacts with Ser 838 to stabilize the activation loop while it interacts with Gln 667 to stabilize the activation loop in open and active state, a similar interaction has been observed for the stabilization of ABL kinase in active conformation. In support, western blotting of FLT-3 autophosphorylation of these mutants, F691L and D835F/Y, from the untreated total cell extracts showed reduced autophosphorylation of FLT3 in comparison with FLT3-ITD (Figure 1c), thus supporting the notion that these mutations confer resistance by destabilizing the active state rather than direct steric hindrance to the drug.

Figure 2

SAR302503 binds to ATP site, an active conformation of the FLT3 kinase. (a) Ribbon depiction of a structural model of FLT3-SAR302503 using the coordinates of the inactive FLT3 kinase (PDB:3CS9), ABL (PDB:2Z60) and JAK2 (PDB:2B7A). Activation loop is shown ...

A recent study has shown the efficacy of Ponatinib on AC220 resistant mutations in F691L/I variant. However, it is ineffective against compound mutations and variants from the activation loop (D835F/Y), suggesting that these variants will become significant clinical challenge.13 Our study demonstrates that the SAR302503 is potently active against these resistant variants and prevents the emergence of resistant clones at Cmax (3 μM) of SAR302503.6, 9 Given the preclinical success of SAR302503 as a well-tolerated oral agent for the treatment of polycythemia vera and Myelofibrosis, its selectivity against tyrosine-kinase inhibitor (TKI) resistant variants of FLT3-ITD supports the clinical evaluation of SAR302503 in TKI-treated as well as TKI naive FLT3-ITD+ AML patients. To that end, SAR302503 may represent an effective first-line TKI therapy in AML patients. In addition, a lack of in vitro resistance against SAR302503 suggests that it will be more effective in managing the clinical resistance.

Activating mutations in FLT3 (Fms-like tyrosine kinase 3) by internal tandem duplication (ITD) mutations are found in approximately 30% of patients with acute myeloid leukemia (AML) and are associated with poor outcome in this patient population. Numerous FLT3 inhibitors have been tested for the treatment of AML, but these inhibitors have shown variable responses that were attributed to heterogeneity in AML and mechanistic differences associated with inhibitory mechanism employed by these inhibitors. 1 Nevertheless, many patients showed a reduction in blast counts and hematological improvements, but these remissions were not durable that questioned the validity of FLT3 as a therapeutic target in AML. To address this, an elegant translational study designed by Neil Shah's group has identified the AC220 resistant mutations in all FLT3-ITD relapsed patients that definitively demonstrated the validity of FLT3-ITD as a therapeutic target in human AML. 2 Furthermore, the emergence of resistant mutations has been reported from the relapsed AML patients with FLT3-ITD treated with PKC412 3 and sorafenib. 4 In addition, an in vitro resistant screening identified mutation at gatekeeper residue conferred cross-resistance to all known FLT3 inhibitors. 5 These observations suggest that secondary mutations conferring resistance in the kinase domain will pose a significant clinical challenge, which prompted us to identify new inhibitors against the FLT3 resistant variants.
SAR302503 is a rationally designed small-molecule ATP-competitive inhibitor of JAK2, and it had been shown to have a high degree of kinase selectivity for JAK2 and FLT3 in in vitro kinase assays. 6,7 Given its equal potency toward FLT3 prompted us to test its efficacy against FLT3-ITD variants resistant to AC220. SAR302503 inhibited proliferation of BAF3 cells expressing FLT3 wild type and FLT3-ITD, with IC 50 values of 119 and 330 nM, respectively (Figure 1a), whereas parental BAF3 cells and BAF3-JAK2-V617F cells were inhibited at IC 50 values of B1100 and 600 nM, respectively (Supplementary Figure 1a). In accordance to this, western blotting of phospho-STAT5 and phospho-FLT3 showed reduced phosphorylation at concentrations that parallel the concentrations required to inhibit cell proliferation (Figure 1c). These observations suggest that SAR302503 is more selective to FLT3-WT4FLT3-ITD4JAK2-V617F, and it can be exploited for therapeutic targeting of FLT3-ITD in AML (Supplementary Figure 1a). Next, we analyzed the activity of SAR302503 against five different kinase domain variants of FLT3-ITD that had been shown to confer resistance against AC220. The growth of BAF3 cells expressing resistant variants of FLT3-ITD was inhibited completely within the range of 800 nM ( Figure 1a). Interestingly, two FLT3-ITD variants (D839G and Y842H) were found to be hypersensitive to the drug (Figures 1a and b) while mutations at gatekeeper residue F691L and D835F/Y showed only twofold resistance to the drug ( Figure 1b). Next, we performed in vitro resistant screening as described previously, 8 to identify patterns of drug resistance using cells expressing FLT3-ITD, FLT3-ITD-F691L and FLT3-ITD-D835Y at 3 mM (C max value in human 6,9 ) of SAR302503. Surprisingly, we could not see the emergence of resistant clones against SAR302503 at this concentration (Supplementary Figure 1b). To rule out any off-target effect, we performed screening of BAF3 cells expressing Tel-JAK3 (having a cellular IC 50 value of 2300 nm) at 2000 and 3000 nm of SAR302503, which showed growth of innumerable colonies (Supplementary Figure 1b). These observations suggest that the SAR302503 is a potent inhibitor of FLT3-ITD and is very effective against AC220 resistant variants and prevents the emergence of resistant clones.
To understand the structural basis of SAR302503-mediated inhibition of FLT3 kinase, we constructed a model of the SAR302503-FLT3 kinase structure using a combination of homology modeling and induced-fit docking. 10 We used the crystal structure of FLT3 crystallized in an inactive conformation and with related inhibitor À kinase complexes: for example, ABL complexed with nilotinib in closed and inactive conformation (PDB:3CS9); Ppy-A bound to the active conformation of ABL-T315I (PDB:2Z60); and JAK2 bound to isoquinoline in active conformation (PDB:2B7A). Our in silico analysis predicts that SAR302503 binds to an open and enzymatically active conformation of FLT3, in which residue Phe 830 of the DFG motif is displaced to accommodate the benzensulfonamide ring of the inhibitor (Figure 2). In this model, the pyrimidine ring of SAR302503 makes hydrophobic contacts with Leu 616 and Leu 818. Similar to other ATP-competitive inhibitors, SAR302503 anchors to the ATP site by two hydrogen bonds with residues Cys 694 from the kinase hinge region and Asn 816 from the catalytic loop (Figures 2a and b). Our model suggests that the enhanced sensitivity of SAR302503 for the variants D839G and Y842H is due to the destabilization of the inactive conformation by these mutations that in effect would stabilize the open and active conformation to which the drug preferentially binds, thus conferring hypersensitive response (Figures 2d and e). Given the lack of direct interaction of SAR302503 with resistant variants F691L and D835F/Y, our model suggests that these mutations would destabilize the open and active conformation rather than inactivating the kinase, therefore having sufficient catalytic activity to support cellular transformation. In support of this model, a substitution of Leucine for Phe 691 will weaken the hydrophobic spine that in effect will destabilize the active conformation and may weaken the kinase. 11 However, substitution of Asp 835 with phenylalanine will can bind only in DFG-in conformational state (residues shown in green sticks), in which phenylalanine of the DFG motif is aligned in such a way that it coordinates with hydrophobic spine residues to stabilize the active state, a common feature in all kinases suggesting that SAR302503 can only bind to active state and thus can be classified as a type I inhibitor. (c) Active site showing the steric clash of the benzene sulfonamide group of SAR302503 with Phe 830 in DGF-out conformation (F830 from the DFG motif shown in surface). (d) Structure of autoinhibited FLT3 shown in ribbon with modeled activation loops in active conformation (shown in green) and in inactive conformation (shown in red). The juxtamembrane domain (orange) nearly spans the length of the kinase molecule and seemingly displaces the activation loop from the active state to favor inactive conformation. Amino-acid residues that were mutated to confer drug resistance are shown as sticks. (e) Conformation of activation loops in active (green) and inactive (red) states showing interaction of Y842 with D829 (DFG motif ) through R834 and D839 with N934 to stabilize the inactive conformation, and thus mutation at these sites may favor or stabilize active state that will be more sensitive to inhibition by type I inhibitors. In support of this model, variant Y842H showing increased autophosphorylation in untreated sample, whereas D839G did not show significant change suggesting that it only destabilizes the inactive conformation and may be unable to stabilize the fully active conformation as we have observed in the Y842H. Nonetheless, it supports that mutant D839G stabilizes a conformation favoring efficient SAR302503 binding that reflects in hypersensitive response. The hypersensitive response of variant D839G is less than Y842H suggesting that the later is stabilizing a fully active state that binds the drug more efficiently. Nevertheless, these speculations require detail structural studies for better understanding and developing next-generation FLT3 inhibitors.
Letter to the Editor destabilize the activation loop from both active and inactive conformation suggesting that it is stabilizing an intermediate conformational state. 12 In inactive conformation, Asp 835 interacts with Ser 838 to stabilize the activation loop while it interacts with Gln 667 to stabilize the activation loop in open and active state, a similar interaction has been observed for the stabilization of ABL kinase in active conformation. In support, western blotting of FLT-3 autophosphorylation of these mutants, F691L and D835F/Y, from the untreated total cell extracts showed reduced autophosphorylation of FLT3 in comparison with FLT3-ITD (Figure 1c), thus supporting the notion that these mutations confer resistance by destabilizing the active state rather than direct steric hindrance to the drug.
A recent study has shown the efficacy of Ponatinib on AC220 resistant mutations in F691L/I variant. However, it is ineffective against compound mutations and variants from the activation loop (D835F/Y), suggesting that these variants will become significant clinical challenge. 13 Our study demonstrates that the SAR302503 is potently active against these resistant variants and prevents the emergence of resistant clones at C max (3 mM) of SAR302503. 6,9 Given the preclinical success of SAR302503 as a well-tolerated oral agent for the treatment of polycythemia vera and Myelofibrosis, its selectivity against tyrosine-kinase inhibitor (TKI) resistant variants of FLT3-ITD supports the clinical evaluation of SAR302503 in TKI-treated as well as TKI naive FLT3-ITD þ AML patients. To that end, SAR302503 may represent an effective first-line TKI therapy in AML patients. In addition, a lack of in vitro resistance against SAR302503 suggests that it will be more effective in managing the clinical resistance.