FDA Approval Summary: Alpelisib Plus Fulvestrant for Patients with HR-positive, HER2-negative, PIK3CA-mutated, Advanced or Metastatic Breast Cancer.

On May 24, 2019, the FDA granted regular approval to alpelisib in combination with fulvestrant for postmenopausal women, and men, with hormone receptor (HR)-positive, HER2-negative, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated, advanced or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen. Approval was based on the SOLAR-1 study, a randomized, double-blind, placebo-controlled trial of alpelisib plus fulvestrant versus placebo plus fulvestrant. The primary endpoint was investigator-assessed progression-free survival (PFS) per RECIST v1.1 in the cohort of trial participants whose tumors had a PIK3CA mutation. The estimated median PFS by investigator assessment in the alpelisib plus fulvestrant arm was 11 months [95% confidence interval (CI), 7.5-14.5] compared with 5.7 months (95% CI, 3.7-7.4) in the placebo plus fulvestrant arm (HR, 0.65; 95% CI, 0.50-0.85; two-sided P = 0.001). The median overall survival was not yet reached for the alpelisib plus fulvestrant arm (95% CI, 28.1-NE) and was 26.9 months (95% CI, 21.9-NE) for the fulvestrant control arm. No PFS benefit was observed in trial participants whose tumors did not have a PIK3CA mutation (HR, 0.85; 95% CI, 0.58-1.25). The most common adverse reactions, including laboratory abnormalities, on the alpelisib plus fulvestrant arm were increased glucose, increased creatinine, diarrhea, rash, decreased lymphocyte count, increased gamma glutamyl transferase, nausea, increased alanine aminotransferase, fatigue, decreased hemoglobin, increased lipase, decreased appetite, stomatitis, vomiting, decreased weight, decreased calcium, decreased glucose, prolonged activated partial thromboplastin time, and alopecia.

Protein delivery using engineered virus-like particles.

Over the years, researchers have developed several methods to deliver macromolecules into the cytosol and nucleus of living cells. However, there are limitations to all of these methods. The problems include (i) inefficient uptake, (ii) endosomal entrapment, (iii) delivery that is restricted to certain cell types, and (iv) damage to cells in the delivery process. Retroviral vectors are often used for gene delivery; however, integration of the genome of retroviral vector into the host genome can have serious consequences. Here we describe a safe alternative in which virus-like particles (VLPs), derived from an avian retrovirus, are used to deliver protein to cells. We show that these VLPs are a highly adaptable platform that can be used to deliver proteins either as part of Gag fusion proteins (intracellular delivery) or on the surface of VLPs. We generated VLPs that contain Gag-Cre recombinase, Gag-Fcy::Fur, and Gag-human caspase-8 as a proof-of-concept and demonstrated that the encapsidated proteins are active in recipient cells. In addition, we show that murine IFN-γ and human TNF-related apoptosis-inducing ligand can be displayed on the surface of VLPs, and that these modified VLPs can cause the appropriate response in cells, as evidenced by phosphorylation of STAT1 and induction of cell death, respectively.

Optimization of a cyclic peptide inhibitor of Ser/Thr phosphatase PPM1D (Wip1).

PPM1D (PP2Cδ or Wip1) was identified as a wild-type p53-induced Ser/Thr phosphatase that accumulates after DNA damage and classified into the PP2C family. It dephosphorylates and inactivates several proteins critical for cellular stress responses, including p38 MAPK, p53, and ATM. Furthermore, PPM1D is amplified and/or overexpressed in a number of human cancers. Thus, inhibition of its activity could constitute an important new strategy for therapeutic intervention to halt the progression of several different cancers. Previously, we reported the development of a cyclic thioether peptide with low micromolar inhibitory activity toward PPM1D. Here, we describe important improvements in the inhibitory activity of this class of cyclic peptides and also present a binding model based upon the results. We found that specific interaction of an aromatic ring at the X1 position and negative charge at the X5 and X6 positions significantly increased the inhibitory activity of the cyclic peptide, with the optimized molecule having a K(i) of 110 nM. To the best of our knowledge, this represents the highest inhibitory activity reported for an inhibitor of PPM1D. We further developed an inhibitor selective for PPM1D over PPM1A with a K(i) of 2.9 µM. Optimization of the cyclic peptide and mutagenesis experiments suggest that a highly basic loop unique to PPM1D is related to substrate specificity. We propose a new model for the catalytic site of PPM1D and inhibition by the cyclic peptides that will be useful both for the subsequent design of PPM1D inhibitors and for identification of new substrates.

Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

Tus, an E. coli protein, contains mammalian nuclear targeting and exporting signals.

Shuttling of proteins between nucleus and cytoplasm in mammalian cells is facilitated by the presence of nuclear localization signals (NLS) and nuclear export signals (NES), respectively. However, we have found that Tus, an E. coli replication fork arresting protein, contains separate sequences that function efficiently as NLS and NES in mammalian cell lines, as judged by cellular location of GFP-fusion proteins. The NLS was localized to a short stretch of 9 amino acids in the carboxy-terminus of Tus protein. Alterations of any of these basic amino acids almost completely abolished the nuclear targeting. The NES comprises a cluster of leucine/hydrophobic residues located within 21 amino acids at the amino terminus of Tus. Finally, we have shown that purified GFP-Tus fusion protein or GFP-Tus NLS fusion protein, when added to the culture media, was internalized very efficiently into mammalian cells. Thus, Tus is perhaps the first reported bacterial protein to possess both NLS and NES, and has the capability to transduce protein into mammalian cells.

High-throughput protein expression using cell-free system.

One of the main challenges in this post genomic era is the development and implementation of efficient methods of protein synthesis. A clear understanding of the role of genes in an organism is to comprehend the biological functions of all of its proteins. Acquiring this knowledge will depend in part on the success of rapid synthesis and purification of proteins. The future of structural genomics and functional proteomics depends on the availability of abundantly expressing, soluble proteins in a high-throughput manner. Conventional cell based methods of protein expression is rather laborious, time consuming and the ways to fail are numerous including solubility, toxicity to the host and instability (e.g. proteolysis). Cell-free or in vitro protein synthesis, on the other hand allows the expression and analysis of protein synthesis, may solve many of these problems. It is a simple open system which lends itself for manipulations and modifications to influence protein folding, disulfide bond formation, incorporation of unnatural amino acids, protein stability (by incorporating protease inhibitors in the system) and even the expression of toxic proteins. Cell-free synthesis can also be used as a reliable screening methodology for subsequent protein expression in vivo. Furthermore, this technology is readily amenable to automation. Here, we present a protocol for expressing recombinant proteins with high yield in a standard 96-well plate format using E. coli cell-free extract in a batch mode.

Protein microarray on-demand: a novel protein microarray system.

We describe a novel, simple and low-cost protein microarray strategy wherein the microarrays are generated by printing expression ready plasmid DNAs onto slides that can be converted into protein arrays on-demand. The printed expression plasmids serve dual purposes as they not only direct the synthesis of the protein of interest; they also serve to capture the newly synthesized proteins through a high affinity DNA-protein interaction. To accomplish this we have exploited the high-affinity binding (approximately 3-7 x 10 (-13) M) of E. coli Tus protein to Ter, a 20 bp DNA sequence involved in the regulation of E. coli DNA replication. In our system, each protein of interest is synthesized as a Tus fusion protein and each expression construct directing the protein synthesis contains embedded Ter DNA sequence. The embedded Ter sequence functions as a capture reagent for the newly synthesized Tus fusion protein. This "all DNA" microarray can be converted to a protein microarray on-demand without need for any additional capture reagent.

The Wip1 phosphatase PPM1D dephosphorylates SQ/TQ motifs in checkpoint substrates phosphorylated by PI3K-like kinases.

The wild-type p53-induced phosphatase Wip1 (PP2Cdelta or PPM1D) is a member of the protein phosphatase 2C (PP2C) family and controls cell cycle checkpoints in response to DNA damage. p38 MAPK and ATM were identified as physiological substrates of Wip1, and we previously reported a substrate motif that was defined using variants of the p38(180pT 182pY) diphosphorylated peptide, TDDEMpTGpYVAT. However, the substrate recognition motifs for Wip1 have not been fully defined as the sequences surrounding the targeted residues in ATM and p38 MAPK appear to be unrelated. Using a recombinant human Wip1 catalytic domain (rWip1), in this study we measured the kinetic parameters for variants of the ATM(1981pS) phosphopeptide, AFEEGpSQSTTI. We found that rWip1 dephosphorylates phosphoserine and phosphothreonine in the p(S/T)Q motif, which is an essential requirement for substrate recognition. In addition, acidic, hydrophobic, or aromatic amino acids surrounding the p(S/T)Q sequence have a positive influence, while basic amino acids have a negative influence on substrate dephosphorylation. The kinetic constants allow discrimination between true substrates and nonsubstrates of Wip1, and we identified several new putative substrates that include HDM2, SMC1A, ATR, and Wip1 itself. A three-dimensional molecular model of Wip1 with a bound substrate peptide and site-directed mutagenesis analyses suggested that the important residues for ATM(1981pS) substrate recognition are similar but not identical to those for the p38(180pT 182pY) substrate. Results from this study should be useful for predicting new physiological substrates that may be regulated by Wip1 and for developing selective anticancer drugs.

Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids.

To investigate how structural changes in the amino acid side chain affect nucleotide substrate selection in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a variety of non-natural tyrosine analogues were substituted for Tyr115 of p66 RT. RT variants containing meta-Tyr, nor-Tyr, aminomethyl-Phe, and 1- and 2-naphthyl-Tyr were produced in an Escherichia coli coupled transcription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RT and purified by affinity chromatography. Each modified enzyme retained DNA polymerase activity following this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT and was resistant to the chain terminator (-)-beta-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) when examined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTP at low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containing these side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds through the minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogen bonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1 RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain, necessitating rearrangement of the active site.

Enhancement of soluble protein expression through the use of fusion tags.

The soluble expression of heterologous proteins in Escherichia coli remains a serious bottleneck in protein production. Although alteration of expression conditions can sometimes solve the problem, the best available tools to date have been fusion tags that enhance the solubility of expressed proteins. However, a systematic analysis of the utility of these solubility fusions has been difficult, and it appears that many proteins react differently to the presence of different solubility tags. The advent of high-throughput structural genomics programs and advances in cloning and expression technology afford us a new way to compare the effectiveness of solubility tags. This data should allow us to better predict the effectiveness of tags currently in use, and might also provide the information needed to identify new fusion tags.

Enhanced soluble protein expression using two new fusion tags.

Production of soluble recombinant proteins is vital for structure-function analysis and therapeutic applications. Unfortunately, when expressed in a heterologous host, such as Escherichia coli, most proteins are expressed as insoluble aggregates. Two new fusion partners have been identified to address these solubility problems. One of the tags was derived from a bacteriophage T7 protein kinase and the other one from a small E. coli chaperone, Skp. We have expressed a panel of insoluble human proteins including Hif1alpha, IL13, and folliculin as fusion proteins using these tags. Most of these fusion proteins were able to be expressed in a soluble form and could be purified by virtue of a Strep-tag II installed at the amino-terminal end of the fusion partners. In addition, we show that some of these proteins remained soluble after removal of the fusion tags by a site-specific protease. The results with these tags compare favorably to results with the most commonly used solubility tags described in the literature. Therefore, these two new fusion tags have the potential to express soluble proteins when fused with many recalcitrant proteins.

Substrate specificity of the human protein phosphatase 2Cdelta, Wip1.

Wip1, the wild-type p53-induced phosphatase, selectively dephosphorylates a threonine residue on p38 MAPK and mediates a negative feedback loop of the p38 MAPK-p53 signaling pathway. To identify the substrate specificity of Wip1, we prepared a recombinant human Wip1 catalytic domain (rWip1) and measured kinetic parameters for phosphopeptides containing the dephosphorylation sites in p38alpha and in a new substrate, UNG2. rWip1 showed properties that were comparable to those of PP2Calpha or full-length Wip1 in terms of affinity for Mg(2+), insensitivity to okadaic acid, and threonine dephosphorylation. The substrate specificity constant k(cat)/K(m) for a diphosphorylated peptide with a pTXpY sequence was 6-8-fold higher than that of a monophosphorylated peptide with a pTXY sequence, while PP2Calpha showed a preference for monophosphorylated peptides. Although individual side chains before and after the pTXpY sequence of the substrate did not have a significant effect on rWip1 activity, a chain length of at least five residues, including the pTXpY sequence, was important for substrate recognition by rWip1. Moreover, the X residue in the pTXpY sequence affected affinity for rWip1 and correlated with selectivity for MAPKs. These findings suggest that substrate recognition by Wip1 is centered toward a very narrow region around the pTXpY sequence. Three-dimension homology models of Wip1 with bound substrate peptides were constructed, and site-directed mutagenesis was performed to confirm the importance of specific residues for substrate recognition. The results of our study should be useful for predicting new physiological substrates and for designing specific Wip1 inhibitors.

Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase.

A highly efficient cell-free translation system has been combined with suppressor tRNA technology to substitute nor-Tyr and 3-fluoro-Tyr in place of Tyr183 at the DNA polymerase active site of p66 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). Supplementing the wild-type HIV-1 p51 RT subunit into this translation system permitted reconstitution of the biologically relevant p66/p51 heterodimer harboring Tyr analogs exclusively on the catalytically competent p66 subunit. Addition of an affinity tag at the p66 C-terminus allowed rapid, one-step purification of reconstituted and selectively mutated heterodimer HIV-1 RT via strep-Tactin-agarose affinity chromatography. The purified enzyme was demonstrated to be free of contaminating nucleases, allowing characterization of the DNA polymerase and ribonuclease H activities associated with HIV-1 RT. Preliminary characterization of HIV-1 RT(nor-Tyr) and HIV-1 RT(m-fluoro-Tyr) is presented. The success of this strategy will facilitate detailed molecular analysis of structurally and catalytically critical amino acids via their replacement with closely related, unnatural analogs.

A novel cell-free protein synthesis system.

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.

Mutant Thermotoga neapolitana DNA polymerase I: altered catalytic properties for non-templated nucleotide addition and incorporation of correct nucleotides.

Thermotoga neapolitana (Tne) DNA polymerase belongs to the DNA polymerase I (Pol I) family. The O-helix region of these polymerases is involved in dNTP binding and also plays a role in binding primer-template during DNA synthesis. Here we report that mutations in the O-helix region of Tne DNA polymerase (Arg722 to His, Tyr or Lys) almost completely abolished the enzyme's ability to catalyze the template-independent addition of a single base at the 3'-end of newly synthesized DNA in vitro. The mutations did not significantly affect the DNA polymerase catalytic activity and reduced base misinsertions 5- to 50-fold. The same Arg722 mutations dramatically increased the ability of the enzyme's 3'-->5' exonuclease to remove mispaired 3' bases in a primer extension assay. These mutant DNA polymerases can be used to accurately amplify target DNA in vitro for gene cloning and genotyping analysis.

The role of template-primer in protection of reverse transcriptase from thermal inactivation.

We compared the thermal stabilities of wild-type recombinant avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) with those of mutants of the recombinant enzymes lacking RNase H activity. They differed in resistance to thermal inactivation at elevated temperatures in the presence of an RNA/DNA template-primer. RNase H-minus RTs retained the ability to efficiently synthesize cDNA at much higher temperatures. We show that the structure of the template-primer has a critical bearing on protection of RT from thermal inactivation. RT RNase H activity rapidly alters the structure of the template-primer to forms less tightly bound by RT and thus less able to protect the enzyme at elevated temperatures. We also found that when comparing wild-type or mutant AMV RT with the respective M-MLV RT, the avian enzymes retained more DNA synthetic activity at elevated temperatures than murine RTs. Enzyme, template-primer interaction again played the most significant role in producing these differences. AMV RT binds much tighter to template- primer and has a much greater tendency to remain bound during cDNA synthesis than M-MLV RT and therefore is better protected from heat inactivation.