Peptide Drug Conjugates and Their Role in Cancer Therapy.

Drug conjugates have become a significant focus of research in the field of targeted medicine for cancer treatments. Peptide-drug conjugates (PDCs), a subset of drug conjugates, are composed of carrier peptides ranging from 5 to 30 amino acid residues, toxic payloads, and linkers that connect the payload to the peptide. PDCs are further broken down into cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs), each having their own differences in the delivery of cytotoxic payloads. Generally, PDCs as compared to other drug conjugates-like antibody-drug conjugates (ADCs)-have advantages in tumor penetration, ease of synthesis and cost, and reduced off-target effects. Further, as compared to traditional cancer treatments (e.g., chemotherapy and radiation), PDCs have higher specificity for the target cancer with generally less toxic side effects in smaller doses. However, PDCs can have disadvantages such as poor stability and rapid renal clearance due to their smaller size and limited oral bioavailability due to digestion of its peptide structure. Some of these challenges can be overcome with modifications, and despite drawbacks, the intrinsic small size of PDCs with high target specificity still makes them an attractive area of research for cancer treatments.

Structural analysis of a type 1 ribosome inactivating protein reveals multiple L­asparagine­N­acetyl­D­glucosamine monosaccharide modifications: Implications for cytotoxicity.

Pokeweed antiviral protein (PAP) belongs to the family of type I ribosome­inactivating proteins (RIPs): Ribotoxins, which function by depurinating the sarcin­ricin loop of ribosomal RNA. In addition to its antibacterial and antifungal properties, PAP has shown promise in antiviral and targeted tumor therapy owing to its ability to depurinate viral RNA and eukaryotic rRNA. Several PAP genes are differentially expressed across pokeweed tissues, with natively isolated seed forms of PAP exhibiting the greatest cytotoxicity. To help elucidate the molecular basis of increased cytotoxicity of PAP isoenzymes from seeds, the present study used protein sequencing, mass spectroscopy and X-ray crystallography to determine the complete covalent structure and 1.7 Å X­ray crystal structure of PAP­S1aci isolated from seeds of Asian pokeweed (Phytolacca acinosa). PAP­S1aci shares ~95% sequence identity with PAP­S1 from P. americana and contains the signature catalytic residues of the RIP superfamily, corresponding to Tyr72, Tyr122, Glu175 and Arg178 in PAP­S1aci. A rare proline substitution (Pro174) was identified in the active site of PAP­S1aci, which has no effect on catalytic Glu175 positioning or overall active­site topology, yet appears to come at the expense of strained main­chain geometry at the pre­proline residue Val173. Notably, a rare type of N­glycosylation was detected consisting of N­acetyl­D­glucosamine monosaccharide residues linked to Asn10, Asn44 and Asn255 of PAP­S1aci. Of note, our modeling studies suggested that the ribosome depurination activity of seed PAPs would be adversely affected by the N­glycosylation of Asn44 and Asn255 with larger and more typical oligosaccharide chains, as they would shield the rRNA­binding sites on the protein. These results, coupled with evidence gathered from the literature, suggest that this type of minimal N­glycosylation in seed PAPs and other type I seed RIPs may serve to enhance cytotoxicity by exploiting receptor­mediated uptake pathways of seed predators while preserving ribosome affinity and rRNA recognition.

Structural and functional characterization of Falcipain-2, a hemoglobinase from the malarial parasite Plasmodium falciparum.

Malaria is caused by protozoan erythrocytic parasites of the Plasmodium genus, with Plasmodium falciparum being the most dangerous and widespread disease-causing species. Falcipain-2 (FP-2) of P. falciparum is a papain-family (C1A) cysteine protease that plays an important role in the parasite life cycle by degrading erythrocyte proteins, most notably hemoglobin. Inhibition of FP-2 and its paralogues prevents parasite maturation, suggesting these proteins may be valuable targets for the design of novel antimalarial drugs, but lack of structural knowledge has impeded progress toward the rational discovery of potent, selective, and efficacious inhibitors. As a first step toward this goal, we present here the crystal structure of mature FP-2 at 3.1 A resolution, revealing novel structural features of the FP-2 subfamily proteases including a dynamic beta-hairpin hemoglobin binding motif, a flexible N-terminal alpha-helical extension, and a unique active-site cleft. We also demonstrate by biochemical methods that mature FP-2 can proteolytically process its own precursor in trans at neutral to weakly alkaline pH, that the binding of hemoglobin to FP-2 is strictly pH-dependent, and that FP-2 preferentially binds methemoglobin over hemoglobin. Because the specificity and proteolytic activity of FP-2 toward its multiple targets appears to be pH-dependent, we suggest that environmental pH may play an important role in orchestrating FP-2 function over the different life stages of the parasite. Moreover, it appears that selectivity of FP-2 for methemoglobin may represent an evolutionary adaptation to oxidative stress conditions within the host cell.

Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected].

Enzymes of the Rel/Spo family enable bacteria to survive prolonged periods of nutrient limitation by producing an intracellular signaling alarmone, (p)ppGpp, which triggers the so-called stringent response. Both the synthesis of (p)ppGpp from ATP and GDP(GTP), and its hydrolysis to GDP(GTP) and pyrophosphate, are catalyzed by Rel/Spo proteins. The 2.1 A crystal structure of the bifunctional catalytic fragment of the Rel/Spo homolog from Streptococcus dysgalactiae subsp. equisimilis, Rel(Seq), reveals two conformations of the enzyme corresponding to known reciprocal activity states: (p)ppGpp-hydrolase-OFF/(p)ppGpp-synthetase-ON and hydrolase-ON/synthetase-OFF. The hydrolase and synthetase domains bear remarkable similarities to the catalytic domains of the cyclic phosphodiesterase and nucleotidyltransferase superfamilies, respectively. The active sites, separated by more than 30 A, contain bound nucleotides including an unusual (p)ppGpp derivative, GDP-2':3'-cyclic monophosphate. Reciprocal regulation of the antagonistic catalytic activities, suggested by the structure, is supported by mutagenesis experiments and appears to involve ligand-induced signal transmission between the two active sites.

Sugar-mediated lattice contacts in crystals of a plant glycoprotein.

Pokeweed antiviral protein, PAP-S(aci), isolated from seeds of the Chinese pokeweed plant, Phytolacca acinosa, belongs to the family of type-1 ribosome-inactivating proteins (RIPs). Type-1 RIPs are approximately 30-kDa N-glycosidases that inactivate eukaryotic and prokaryotic ribosomes via a site-specific depurination of ribosomal RNA (rRNA). Here we describe the preliminary X-ray structure determination at 1.7 A resolution of one PAP isoenzyme from seeds, PAP-S1(aci), after crystallisation from a heterogeneous mixture of two isoenzymes. PAP-S1(aci) possesses a rare type of glycosylation, specifically, N-linked N-acetyl-D-glucosamine monosaccharide (GlcNAc) substitutions at canonical Asn-Xaa-Ser/Thr sequons. One GlcNAc residue was found to play a critical role in crystal lattice formation, forming a packing interface across a crystallographic two-fold with the identical sequon of an adjacent monomer. This observation suggests that deglycosylation protocols for the crystallisation of glycoproteins should be designed to allow for exploitation of the crystal packing potential of the innermost core sugar residue (N-linked GlcNAc or O-linked GalNAc).