Kazuya Shimura

Broad Antiretroviral Activity and Resistance Profile of the Novel Human Immunodeficiency Virus Integrase Inhibitor Elvitegravir (JTK-303/GS-9137)

ABSTRACT Integrase (IN), an essential enzyme of human immunodeficiency virus (HIV), is an attractive antiretroviral drug target. The antiviral activity and resistance profile in vitro of a novel IN inhibitor, elvitegravir (EVG) (also known as JTK-303/GS-9137), currently being developed for the treatment of HIV-1 infection are described. EVG blocked the integration of HIV-1 cDNA through the inhibition of DNA strand transfer. EVG inhibited the replication of HIV-1, including various subtypes and multiple-drug-resistant clinical isolates, and HIV-2 strains with a 50% effective concentration in the subnanomolar to nanomolar range. EVG-resistant variants were selected in two independent inductions, and a total of 8 amino acid substitutions in the catalytic core domain of IN were observed. Among the observed IN mutations, T66I and E92Q substitutions mainly contributed to EVG resistance. These two primary resistance mutations are located in the active site, and other secondary mutations identified are proximal to these primary mutations. The EVG-selected IN mutations, some of which represent novel IN inhibitor resistance mutations, conferred reduced susceptibility to other IN inhibitors, suggesting that a common mechanism is involved in resistance and potential cross-resistance. The replication capacity of EVG-resistant variants was significantly reduced relative to both wild-type virus and other IN inhibitor-resistant variants selected by L-870,810. EVG and L-870,810 both inhibited the replication of murine leukemia virus and simian immunodeficiency virus, suggesting that IN inhibitors bind to a conformationally conserved region of various retroviral IN enzymes and are an ideal drug for a range of retroviral infections.

Elvitegravir: a new HIV integrase inhibitor.

Integration is a distinctive and essential process in the HIV infection cycle and thus represents an attractive antiviral drug target. Integrase inhibitors combined with other classes of drug might contribute to long-lasting suppression of HIV type-1 (HIV-1) replication for many patients. Of the numerous potential integrase inhibitor leads that have been reported, few have reached clinical trials and only one, raltegravir, has been approved (in late 2007) for the treatment of HIV-1-infected patients. Another integrase inhibitor, elvitegravir, is currently showing promise in Phase III clinical studies. Once-daily administration of elvitegravir has a comparable antiviral activity to twice-daily of raltegravir in HIV-1-infected patients. Here, we highlight the salient features of elvitegravir: its chemical structure compared with representative integrase inhibitors, mechanism of action, in vitro and in vivo activity against HIV and other retroviruses, and the effect of integrase polymorphisms and resistance mutations on its anti-HIV activity.

CXCR4 Stimulates Macropinocytosis: Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV

CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.

Design of Peptide-based Inhibitors for Human Immunodeficiency Virus Type 1 Strains Resistant to T-20

Enfuvirtide (T-20) is a fusion inhibitor that suppresses replication of human immunodeficiency virus (HIV) variants with multi-drug resistance to reverse transcriptase and protease inhibitors. It is a peptide derived from the C-terminal heptad repeat (C-HR) of HIV-1 gp41, and it prevents interactions between the C-HR and the N-terminal HR (N-HR) of gp41, thus interfering with conformational changes that are required for viral fusion. However, prolonged therapies with T-20 result in the emergence of T-20-resistant strains that contain primary mutations such as N43D in the N-HR of gp41 (where T-20 and C-HR bind) that help the virus escape at a fitness cost. Such variants often go on to acquire a secondary mutation, S138A, in the C-HR of gp41 region that corresponds to the sequence of T-20. We demonstrate here that the role of S138A is to compensate for the impaired fusion kinetics of HIV-1s carrying primary mutations that abrogate binding of T-20. To preempt this escape strategy, we designed a modified T-20 variant containing the S138A substitution and showed that it is a potent inhibitor of both T-20-sensitive and T-20-resistant viruses. Circular dichroism analysis revealed that the S138A provided increased stability of the 6-helix bundle. We validated our approach on another fusion inhibitor, C34. In this case, we designed a variant of C34 with the secondary escape mutation N126K and showed that it can effectively inhibit replication of C34-resistant HIV-1. These results prove that it is possible to design improved peptide-based fusion inhibitors that are efficient against a major mechanism of drug resistance.

Resistance profiles of novel electrostatically constrained HIV-1 fusion inhibitors.

Human immunodeficiency virus (HIV) gp41 plays a key role in viral fusion; the N- and C-terminal heptad repeats (N-HR and C-HR) of gp41 form a stable 6-helical conformation for fusion. Therefore, HR-derived peptides, such as enfuvirtide (T-20), inhibit HIV-1 fusion by acting as decoys, and have been used for the treatment of HIV-1 infection. However, the efficacy of T-20 is attenuated by resistance mutations in gp41, including V38A and N43D. To suppress the resistant variants, we previously developed electrostatically constrained peptides, SC34 and SC34EK, and showed that both exhibited potent anti-HIV-1 activity against wild-type and T-20-resistant variants. In this study, to clarify the resistance mechanism to this next generation of fusion inhibitors, we selected variants with resistance to SC34 and SC34EK in vitro. The resistant variants had multiple mutations in gp41. All of these mutations individually caused less than 6-fold resistance to SC34 and SC34EK, indicating that there is a significant genetic barrier for high-level resistance. Cross-resistance to SC34 and SC34EK was reduced by a simple difference in the polarity of two intramolecular electrostatic pairs. Furthermore, the selected mutations enhanced the physicochemical interactions with N-HR variants and restored activities of the parental peptide, C34, even to resistant variants. These results demonstrate that our approach of designing gp41-binding inhibitors using electrostatic constraints and information derived from resistance studies produces inhibitors with enhanced activity, high genetic barrier, and distinct resistance profile from T-20 and other inhibitors. Hence, this is a promising approach for the design of future generation peptide fusion inhibitors.

Potent CXCR4 Antagonists Containing Amidine Type Peptide Bond Isosteres

A series of FC131 [cyclo(-d-Tyr-Arg-Arg-Nal-Gly-)] analogues containing amidine type peptide bond isosteres were synthesized as selective CXC chemokine receptor type 4 (CXCR4) antagonists. An isosteric amidine substructure was constructed by a macrocyclization process using nitrile oxide-mediated C-N bond formation. All of the amidine-containing FC131 analogues exhibited potent SDF-1 binding inhibition to CXCR4. The Nal-Gly-substituted analogue was characterized as one of the most potent cyclic pentapeptide-based CXCR4 antagonists reported to date. The improved activity against human immunodeficiency virus (HIV) type-1 X4 strains suggested that addition of another basic amidine group to the peptide backbone effectively increases the selective binding of the peptides to CXCR4 receptor.

Structure-activity relationship study of pyrimido[1,2-c][1,3]benzothiazin-6-imine derivatives for potent anti-HIV agents.

3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) is an antiretroviral agent with submicromolar inhibitory activity against human immunodeficiency virus-1 (HIV-1) and HIV-2 infection. In the current study, the structure-activity relationships of accessory groups at the 3- and 9-positions of pyrimido[1,2-c][1,3]benzothiazin-6-imine were investigated for the development of more potent anti-HIV agents. Several different derivatives containing a 9-aryl group were designed and synthesized using Suzuki-Miyaura cross-coupling and Ullmann coupling reactions. Modification of the m-methoxyphenyl or benzo[d][1,3]dioxol-5-yl group resulted in improved anti-HIV activity. In addition, the 2,4-diazaspiro[5.5]undec-2-ene-fused benzo[e][1,3]thiazine derivatives were designed and tested for their anti-HIV activities. The most potent 9-(benzo[d][1,3]dioxol-5-yl) derivative was two-threefold more effective against several strains of HIV-1 and HIV-2 than the parent compound, PD 404182.

Synonymous mutations in stem-loop III of Rev responsive elements enhance HIV-1 replication impaired by primary mutations for resistance to enfuvirtide

Primary mutations in HIV-1 that are directly involved in the resistance to enfuvirtide have been well documented. However, secondary mutations that are associated with primary mutations and contribute little to the resistance still remain to be elucidated. This study reveals that synonymous mutations at gp41 Q41 (CAG to CAA) or L44 (UUG to CUG) act as secondary mutations. Complementary mutations in the nucleotide level are located in the Rev responsive element (RRE) of the HIV-1 RNA-genome and maintain the replication kinetics of HIV-1 through increasing the structural stability of stem-loop III in the RRE. Therefore, synonymous mutations in the gp41/RRE sequence improve the viral replication impaired by the primary mutations and play a key role as secondary (complementary) mutations.

Characterization of HIV-1 resistance to a fusion inhibitor, N36, derived from the gp41 amino-terminal heptad repeat

A transmembrane glycoprotein of HIV-1, gp41, plays a central role in membrane fusion of HIV-1 and host cells. Peptides derived from the amino- and carboxyl-terminal heptad repeat (N-HR and C-HR, respectively) of gp41 inhibit this fusion. The mechanism of resistance to enfuvirtide, a C-HR-derived peptide, is well defined; however the mechanism of resistance to N-HR-derived peptides remains unclear. We characterized an HIV-1 isolate resistant to the N-HR-derived peptide, N36. This HIV-1 acquired a total of four amino acid substitutions, D36G, N126K and E137Q in gp41, and P183Q in gp120. Among these substitutions, N126K and/or E137Q conferred resistance to not only N36, but also C34, which is the corresponding C-HR-derived peptide fusion inhibitor. We performed crystallographic and biochemical analysis of the 6-helix bundle formed by synthetic gp41-derived peptides containing the N126K/E137Q substitutions. The structure of the 6-helix bundle with N126K/E137Q was identical to that in wild-type HIV-1 except for the presence of a new hydrogen bond. Denaturing experiments revealed that the stability of the 6-helix bundle of N126K/E137Q is greater than in the wild-type. These results suggest that the stabilizing effect of N126K/E137Q provides resistance to N36 and C34.

Design and synthesis of biotin- or alkyne-conjugated photoaffinity probes for studying the target molecules of PD 404182.

To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on previous structure-activity relationship studies. These probes retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.